Passive immunization for staphylococcus infections

ABSTRACT

Disclosed herein are monoclonal antibodies or binding portion thereof that bind specifically to a Staphylococcus spp. autolysin N-acetylmuramoyl-L-alanine amidase catalytic domain and/or cell wall binding domain, as well as pharmaceutical compositions containing the same. Cell lines expressing the monoclonal antibodies, including hybridomas, are also disclosed. Methods of using the monoclonal antibodies for installation of orthopedic implants, grafts or medical devices, treating or preventing a Staphylococcus infection, and treating osteomyelitis are described, as are diagnostic methods for the detection of Staphylococcus in a sample.

This application is a divisional of U.S. patent application Ser. No.15/104,104, which is a national stage application under 35 U.S.C. § 371of PCT Application No. PCT/US2014/070337, filed Dec. 15, 2014, whichclaims the priority benefit of U.S. Provisional Patent Application Ser.No. 61/915,953, filed Dec. 13, 2013, which is hereby incorporated byreference in its entirety.

FIELD OF USE

Disclosed herein are methods and compositions for the passiveimmunization against Staphylococcus infection, particularly for theprevention or treatment of osteomyelitis and for infections arising fromimplantation of a medical device, or an orthopedic implant or graft.Antibodies that bind specifically to a Staphylococcus spp. autolysinN-acetylmuramoyl-L-alanine amidase catalytic domain and/or cell wallbinding domain, and pharmaceutical compositions containing the same canbe used for these purposes.

BACKGROUND

There is a great need for novel interventions of chronic osteomyelitis(OM) as approximately 112,000 orthopedic device-related infections occurper year in the US, at an approximate hospital cost of $15,000-70,000per incident (Darouiche, “Treatment of Infections Associated WithSurgical Implants,” N. Engl. J. Med. 350(14):1422-9 (2004)). Althoughimprovements in surgical technique and aggressive antibiotic prophylaxishave decreased the infection rate following orthopedic implant surgeryto 1-5%, osteomyelitis (OM) remains a serious problem and appears to beon the rise from minimally invasive surgery (Mahomed et al., “Rates andOutcomes of Primary and Revision Total Hip Replacement in the UnitedStates Medicare Population,” J. Bone Joint Surg. Am. 85(A-1):27-32(2003); WHO Global Strategy for Containment of Antimicrobial Resistance,2001). The significance of this resurgence, 80% of which is due toStaphylococcus aureus, is amplified by the fact that ˜50% of clinicalisolates are methicillin resistant S. aureus (MRSA). While the infectionrates for joint prostheses and fracture-fixation devices have been only0.3-11% and 5-15% of cases, respectively, over the last decade (Lew andWaldvogel, “Osteomyelitis,” Lancet 364(9431):369-79 (2004); Toms et al.,“The Management of Peri-Prosthetic Infection in Total JointArthroplasty,” J. Bone Joint Surg. Br. 88(2):149-55 (2006)), this resultmay lead to amputation or death. Additionally, the popularization of“minimally invasive surgery” for elective total joint replacements (TJR)in which the very small incision often leads to complications from theprosthesis contacting skin during implantation, has markedly increasedthe incidence of OM (Mahomed et al., “Rates and Outcomes of Primary andRevision Total Hip Replacement in the United States MedicarePopulation,” J. Bone Joint Surg. Am. 85(A-1):27-32 (2003); WHO GlobalStrategy for Containment of Antimicrobial Resistance, 2001). Theseinfections require a very expensive two-stage revision surgery, andrecent reports suggest that success rates could be as low as 50% (Azzamet al., “Outcome of a Second Two-stage Reimplantation for PeriprostheticKnee Infection,” Clin. Orthop. Relat. Res. 467(7):1706-14 (2009)).However, the greatest concern is the emergence of drug-resistantstaphylococcal strains, most notably MRSA, which has surpassed HIV asthe most deadly pathogen in North America, and continues to make themanagement of chronic OM more difficult and expensive, resulting in agreat demand for novel therapeutic interventions to treat patients withthese infections. There is a great need for alternative interventionalstrategies, particularly for immune-compromised elderly who are theprimary recipients of TJR.

Presently, there are no prophylactic treatments that can protecthigh-risk patients from MRSA, most notably the aging “baby boomers” whoaccount for most of the 1.5 million TJR performed annually in the UnitedStates. A vaccine that would decrease the MRSA incidence by 50-80% wouldnot only reduce the number one complication of joint replacement andopen fracture repair procedures, but also cut the healthcare burden by asimilar amount.

Studies have documented that 80% of chronic OM is caused by S. aureus.These bacteria contain several factors that make them bone pathogensincluding several cell-surface adhesion molecules that facilitate theirbinding to bone matrix (Flock et al., “Cloning and Expression of theGene for a Fibronectin-Binding Protein from Staphylococcus aureus,” EMBOJ. 6(8):2351-7 (1987)), toxins capable of stimulating bone resorption(Nair et al., “Surface-Associated Proteins from Staphylococcus aureusDemonstrate Potent Bone Resorbing Activity,” J. Bone Miner. Res.10(5):726-34 (1995)), and degradation of bone by stimulating increasedosteoclast activity (Marriott et al., “Osteoblasts Express theInflammatory Cytokine Interleukin-6 in a Murine Model of Staphylococcusaureus Osteomyelitis and Infected Human Bone Tissue,” Am. J. Pathol.164(4):1399-406 (2004)). The rate-limiting step in the evolution andpersistence of infection is the formation of biofilm around implanteddevices (Costerton et al., “Bacterial Biofilms: A Common Cause ofPersistent Infections,” Science 284(5418):1318-22 (1999)). Shortly afterimplantation, a conditioning layer composed of host-derivedextracellular matrix components (including fibrinogen, fibronectin, andcollagen) forms on the surface of the implant and invites the adherenceof either free-floating bacteria derived from hematogenous seeding, orbacteria from a contiguous nidus of infection such as from the skinadjacent to a wound, surgical inoculation of bacteria into bone, ortrauma coincident with significant disruption of the associated softtissue bone envelope (Darouiche, “Treatment of Infections AssociatedWith Surgical Implants,” N. Engl. J. Med. 350(14):1422-9 (2004)). Overthe next few days, increased colonial adhesion, bacterial cell division,recruitment of additional planktonic organisms, and secretion ofbacterial extracellular polymeric substances (such as those that formthe glycocalyx) produces a bacterial biofilm. This biofilm serves as adominant barrier to protect the bacteria from the action of antibiotics,phagocytic cells and antibodies and impairs host lymphocyte functions(Gray et al., “Effect of Extracellular Slime Substance fromStaphylococcus epidermidis on the Human Cellular Immune Response,”Lancet 1(8373):365-7 (1984); Johnson et al., “Interference withGranulocyte Function by Staphylococcus epidermidis Slime,” Infect.Immun. 54(1):13-20 (1986); Naylor et al., “Antibiotic Resistance ofBiomaterial-Adherent Coagulase-Negative and Coagulase-PositiveStaphylococci,” Clin. Orthop. Relat. Res. 261:126-33 (1990)).

Another recent discovery is that S. aureus not only colonizes bonematrix, but is also internalized by osteoblasts in vitro (Ellington etal., “Involvement of Mitogen-Activated Protein Kinase Pathways inStaphylococcus aureus Invasion of Normal Osteoblasts,” Infect. Immun.69(9):5235-42 (2001)) and in vivo (Reilly et al., “In VivoInternalization of Staphylococcus aureus by Embryonic ChickOsteoblasts,” Bone 26(1):63-70 (2000)). This provides yet another layerof antibody and antibiotic resistance. This phase of infection occursunder conditions of markedly reduced metabolic activity and sometimesappears as so-called small-colony variants that likely accounts for itspersistence (Proctor et al., “Persistent and Relapsing InfectionsAssociated with Small-Colony Variants of Staphylococcus aureus,” Clin.Infect. Dis. 20(1):95-102 (1995)). At this point the bacteria may alsoexpress phenotypic resistance to antimicrobial treatment, alsoexplaining the high failure rate of short courses of therapy (Chuard etal., “Resistance of Staphylococcus aureus Recovered From InfectedForeign Body in Vivo to Killing by Antimicrobials,” I Infect. Dis.163(6):1369-73 (1991)). Due to these extensive pathogenic mechanism, OMis notorious for its tendency to recur even after years of quiescence,and it is accepted that a complete cure is an unlikely outcome (Maderand Calhoun, “Long-Bone Osteomyelitis Diagnosis and Management,” Hosp.Pract. (Off Ed) 29(10):71-6, 9, 83 passim (1994)).

One of the key questions in the field of chronic OM is why currentknowledge of factors that regulate chronic OM is so limited. Supposedly,the experimental tools necessary to elucidate bacterial virulence geneshave been available for over a century. There are three explanations forthis anomaly. First, although the total number of osteomyelitis cases ishigh, its incidence of 1-5% is too low for rigorous prospective clinicalstudies, with the possible exception of revision arthroplasty. Second,it is well known that in vitro cultures rapidly select for growth oforganisms that do not elaborate an extracellular capsule, such thatbiofilm biology can only be studied with in vivo models (Costerton etal., “Bacterial Biofilms: A Common Cause of Persistent Infections,”Science 284(5418):1318-22 (1999)). This leads to the “greatest obstacle”in this field, which is the absence of a quantitative animal model thatcan assess the initial planktonic growth phase of the bacteria prior tobiofilm formation. To date, much of the knowledge of its pathogenesiscomes from animal models (Norden, “Lessons Learned from Animal Models ofOsteomyelitis,” Rev. Infect. Dis. 10(1):103-10 (1988)), which have beendeveloped for the chicken (Daum et al., “A Model of Staphylococcusaureus Bacteremia, Septic Arthritis, and Osteomyelitis in Chickens,” J.Orthop. Res. 8(6):804-13 (1990)), rat (Rissing et al., “Model ofExperimental Chronic Osteomyelitis in Rats,” Infect. Immun. 47(3):581-6(1985)), guinea pig (Passl et al., “A Model of ExperimentalPost-Traumatic Osteomyelitis in Guinea Pigs,” J. Trauma 24(4):323-6(1984)), rabbit (Worlock et al., “An Experimental Model ofPost-Traumatic Osteomyelitis in Rabbits,” Br. J. Exp. Pathol.69(2):235-44 (1988)), dog (Varshney et al., “Experimental Model ofStaphylococcal Osteomyelitis in Dogs,” Indian J. Exp. Biol. 27(9):816-9(1989)), sheep (Kaarsemaker et al., “New Model for Chronic OsteomyelitisWith Staphylococcus aureus in Sheep,” Clin. Orthop. Relat. Res.339:246-52 (1997)) and most recently mouse (Marriott et al.,“Osteoblasts Express the Inflammatory Cytokine Interleukin-6 in a MurineModel of Staphylococcus aureus Osteomyelitis and Infected Human BoneTissue,” Am. J. Pathol. 164(4):1399-406 (2004)). While these models havebeen used to confirm the importance of bacterial adhesins identifiedfrom in vitro assays (Chuard et al., “Susceptibility of Staphylococcusaureus Growing on Fibronectin-Coated Surfaces to BactericidalAntibiotics,” Antimicrob. Agents Chemother. 37(4):625-32 (1993); Buxtonet al., “Binding of a Staphylococcus aureus Bone Pathogen to Type ICollagen,”Microb. Pathog. 8(6):441-8 (1990); Switalski et al., “ACollagen Receptor on Staphylococcus aureus Strains Isolated FromPatients With Septic Arthritis Mediates Adhesion to Cartilage,” Mol.Microbiol. 7(1):99-107 (1993)), they do not have an outcome measure ofin vivo growth, bacterial load, or osteolysis. Thus, they cannot beefficiently used to assess drug effects, bacterial mutants, and the roleof host factors with transgenic mice.

Based on over 150 years of research, a clear paradigm to explainstaphylococcal pathogenesis has emerged. This model also applies to OM.The initial step of infection occurs when a unicellular bacteriuminvades the body. At this point the microbe must respond toenvironmental changes and express virulence genes that will help itdefeat innate immunity and provide it with adhesin receptors to attachto the host. The bacterium is also dependent on the stochasticavailability of host adhesion targets from necrotic tissue or a foreignbody such as an implant for adherence and surface colonization to occur.Successful completion of these steps leads to an exponential biofilmgrowth phase, which ceases at the point of nutrient exhaustion and/orthe development of adaptive immunity. Following the exponential growthphase the bacteria persist under dormant growth conditions within amultilayered biofilm until quorum sensing-driven changes in geneexpression allow for portions of the biofilm to detach as planktoniccells or mobile segments of biofilm patches (Yarwood, et al., “QuorumSensing in Staphylococcus aureus Biofilms,” J. Bact. 186(6): 1838-1850(2004)). However, at this point the infection is now chronic and cannotbe eradicated by drugs or host immunity. Thus, the focus in this fieldhas been on cell surface extracellular matrix components thatspecifically interact with a class of bacterial adhesins known asMSCRAMMs (microbial surface components recognizing adhesive matrixmolecules) (Patti et al., “MSCRAMM-Mediated Adherence of Microorganismsto Host Tissues,” Annu. Rev. Microbiol. 48:585-617 (1994)). In fact,essentially all anti-S. aureus vaccines developed to date have beendirected against MSCRAMMs that are important for host tissuecolonization and invasion. The goal of these vaccines is to generateantibodies that bind to these bacterial surface antigens, therebyinhibiting their attachment to host tissue and suppressing the biofilmformation which serves as a long term reservoir of infection. Byopsonizing the bacterial surface, these antibodies can also mediate S.aureus clearance by phagocytic cells. Unfortunately, S. aureus has manyadhesins, such that inhibition of one or more may not be sufficient toprevent bacterial attachment. Furthermore, bacterial clearance byphagocytic cells may be limited in avascular tissue such as bone suchthat an antibody alone may need additional anti-microbial mechanisms ofaction to significantly reduce the in vivo planktonic growth of S.aureus and prevent the establishment of chronic OM or reinfection duringrevision total joint replacement surgery.

While PCT Publication Nos. WO2011/140114 and WO2013/066876 to Schwarz etal. describe several monoclonal antibodies (hereinafter “mAbs”) thatbind specifically to Staphylococcus glucosaminidase and inhibit in vivogrowth of a Staphylococcus strain, there remains a need to identifyadditional mAbs that bind specifically to a different Staphylococcustarget and inhibit its function.

The disclosed invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE DISCLOSURE

A first aspect relates to a monoclonal antibody or binding portionthereof that binds specifically to a Staphylococcus spp. autolysinN-acetylmuramoyl-L-alanine amidase catalytic domain or cell wall bindingdomain.

A second aspect relates to a cell line that expresses a monoclonalantibody or binding portion thereof as disclosed herein.

A third aspect relates to a pharmaceutical composition that includes acarrier and one or more monoclonal antibodies or monoclonal antibodybinding portions as disclosed herein.

A fourth aspect relates to a method of introducing an orthopedic implantor medical device into a patient that involves administering to apatient in need of an orthopedic implant an effective amount of amonoclonal antibody or monoclonal antibody binding portion according tothe first aspect as disclosed herein, a pharmaceutical compositionaccording to the third aspect as disclosed herein, or a combinationthereof, and introducing the orthopedic implant, tissue graft, ormedical device into the patient.

A fifth aspect relates to a method of treating or preventing aStaphylococcus infection that includes administering to a patientsusceptible to or having a Staphylococcus infection an effective amountof a monoclonal antibody or monoclonal antibody binding portionaccording to the first aspect as disclosed herein, a pharmaceuticalcomposition according to the third aspect as disclosed herein, or acombination thereof.

A sixth aspect relates to a method of treating osteomyelitis thatinvolves administering to a patient having a Staphylococcus bone orjoint infection an effective amount of a monoclonal antibody ormonoclonal antibody binding portion according to the first aspect asdisclosed herein, a pharmaceutical composition according to the thirdaspect as disclosed herein, or a combination thereof.

A seventh aspect relates to a method of determining the presence ofStaphylococcus in a sample that involves exposing a sample to amonoclonal antibody or binding portion according to the first aspect asdisclosed herein, and detecting whether an immune complex forms betweenthe monoclonal antibody or binding portion and Staphylococcus or aStaphylococcus amidase present in the sample, whereby the presence ofthe immune complex after said exposing indicates that presence ofStaphylococcus in the sample.

Staphylococcus N-acetylmuramoyl-L-alanine amidase (hereinafter “Amd” or“amidase”) has several properties that make it an attractive target forpassive immunization. The amidase is involved in multiple crucial cellfunctions including bacterial cell adhesion, cell division, secretionand biofilm glycocalyx formation through its mediation of autolysiswhich produces glycocalyx extracellular DNA; it is highly conservedamong S. aureus clinical isolates; it is the target of vancomycin and itexpressed throughout the cell cycle. Further, because Amd is displayedon the cell wall, it is accessible to antibodies present in theextracellular milieu.

The monoclonal antibodies and binding portions thereof, as well aspharmaceutical compositions containing the same, are therapeutic agentssuitable for immunotherapy in patients with or at risk for infection byStaphylococcus strains. The power of these monoclonal antibodies isderived from their multiple activities that will hinder growth,adhesion, and immune evasion by Staphylococcus strains. First, asantibodies, they will promote phagocytosis by neutrophils at the site ofincipient Staphylococcus infections. Second, as inhibitors of theStaphylococcus amidase, an enzyme with multiple roles in Staphylococcussurvival and surface colonization, these antibodies potentially hinderone or both of cell division and biofilm formation. Finally, asdemonstrated herein, the disclosed antibodies reduce Staphylococcusspread, as evidenced by the formation of fewer abscesses, and affordmacrophage invasion of abscesses, which promotes the formation ofsterile abscesses and accelerates bone healing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of the domain structure of S. aureusbifunctional autolysin (AtlA), which is representative of allStaphylococcus spp. bifunctional autolysin proteins. Bifunctionalautolysin is synthesized as a 1276 amino acid pre-pro-enzyme. The31-amino acid signal peptide (aa 1-31) is removed during secretion andthe 167-amino acid pro-peptide (aa 32-197) is removed when the autolysinis inserted into the cell wall. After cell division, the matureautolysin is cleaved at amino acid 775 to yield independent AmdR1R2(N-acetylmuramoyl-L-alanine amidase, or amidase (Amd); aa 198-775) andR3Gmd (endo-β-N-acetylglucosaminidase (Gmd); aa 776-1276).

FIG. 2 is a graph showing inhibition of Amd enzymatic activity eightanti-Amd monoclonal antibodies and an isoytpe-matched antibody ofirrelevant specificity. Recombinant Amd (rAmd) was prepared in E. coli(His-AmdR1R2-B in Table 1). rAmd (1.5 μg/mL) was mixed in PBS with aturbid suspension of peptidoglycan prepared from S. aureus cell wallsand its lytic activity was measured by the reduction in turbidity(measured as A₄₉₀) following incubation for 60 minutes at 37° C. (Δ60).For the inhibition test, the concentration of rAmd was sufficient toreduce the A₄₉₀ by 70%. Purified anti-Amd mAbs were added to the rAmd atthe indicated concentrations and then lysis of peptidoglycan by theMab:rAmd mixture was measured. Percent inhibition was calculated as:100×(1−(Δ60A₄₉₀ inhibitor/Δ60A₄₉₀ no inhibitor control)).

FIG. 3 is an image of S. aureus precipitation by representative anti-Amdantibodies. When S. aureus cells are cultured in the presence of mostStaphylococcus-specific mAbs they form into large clusters that fall outof suspension yielding a relatively clear supernatant. USA300LAC S.aureus were cultured in TSB at 37° C. for eight hours in the presence ofthe indicated anti-Amd mAbs, each at 25 μg/mL. The sample containing noantibody (No Ab) and an irrelevant isotype-matched antibody (Isotypecontrol) had turbid supernatants without evident cell pellets; mAbsAmd1.1, Amd1.6, Amd1.8, Amd1.11, and Amd1.16 had clear supernatants andcell pellets. Other mAbs producing clear supernatants and cell pelletsare listed in Table 2, infra, as are mAbs that failed to precipitate S.aureus from suspension.

FIG. 4 illustrates the biomolecular interaction analysis of immobilizedmAb Amd1.6 with soluble Amd. The affinity of the interaction between mAbAmd1.6 and soluble Amd was measured on a Biacore T-200. Rabbitanti-mouse Fc IgG was immobilized on the surface of a CM-5 biosensorchip and used to capture mAb Amd1.6 which then captured Amd from aflowing field. The mass of Amd bound by mAb Amd1.6 is measured inResonance Units (y-axis) against time on the x-axis. The capture(association, t=0 to 120 sec) and release (dissociation, t=120 to 420sec) phases are presented. The experiment was repeated withconcentrations of Amd varying in two-fold increments from 1.56 to 25 nM.Measurements were made according the manufacturer's instructions andkinetic data were analyzed using biomolecular interaction analysis (BIA)evaluation software (version 3.1) from Biacore AB.

FIG. 5 is a graph illustrating the inhibitory effect of anti-Amdantibodies on in vitro biofilm formation as compared to the Amd, Gmd andautolysin deletion mutant strains. A biofilm assay utilizing Calgaryplates was performed by coating the plate and lid pegs with human plasmafor 16 hours at 4° C. S. aureus was then seeded at OD 600 nm of 0.05 inthe presence or absence of 25 μg/mL anti-Amd (Amd1.6), anti-Gmd (1C11)and combination of anti-Amd+anti-Gmd (Amd1.6+1C11) mAbs. Biofilmformation was allowed for 24 hours at 37° C. After washing, biofilmswere stained with crystal violet and biofilm content was measured byspectrophotometry at 595 nm. As a positive control for biofilminhibition, UAMS-1 deficient strain for amidase (Δamd), glucosaminidase(Δgmd) or autolysin (Δatl) were seeded at same OD. Results are reportedas the amount of biofilm formation (i.e., crystal violet staining) as apercentage of the wild type (WT), untreated UAMS-1 culture (A); * p<0.05compared to WT.

FIGS. 6A-E illustrate the effect of passive immunization with anti-Amdmonoclonal antibodies and a combination of anti-Amd and anti-Gmdmonoclonal antibodies on biofilm formation on implants in vivo ascompared to autolysin deficiency. Six-to-ten week old, female Balb/cmice (n≥3) were passively immunized intraperitoneally with anti-Amd(Amd1.6), a combination of anti-Amd and anti-Gmd (Amd1.6+1C11) or an IgGisotype-matched control mAb at a dose of 40 mg/kg. One day later eachmouse was infected with a trans-tibial stainless steel pin contaminatedwith USA300 LAC CA-MRSA strain or its isogenic Δatl mutant. The pinswere left in place to allow the biofilm-based infection to mature. OnDay 14 post-infection the pins were removed and examined by scanningelectron microscopy (SEM). Representative micrographs showing the extentof biofilm formation on the infected implants (pins) are shown: IgGcontrol (FIG. 6A); anti-Amd (Amd1.6, FIG. 6B); anti-Amd+anti-Gmd(Amd1.6+1C11, FIG. 6C); and infected with Δatl mutant (FIG. 6D). Thepercentage of the region of interest (the 0.5×2.0 mm face of the flatpin) covered with biofilm was quantified with NIH software (Image J) andshown in FIG. 6E; * p≤0.05.

FIGS. 7A-C illustrate the effect of passive immunization with anti-Amd,anti-Gmd, and a combination of anti-Amd and anti-Gmd monoclonalantibodies on the reduction in the amount of bone damage. Female Balb/cmice (n=5) were passively immunized with PBS or anti-Gmd (1C11),anti-Amd (1.6) or a combination (1C11+1.6) at a 40 mg/kg dose i.p. aspreviously described (Varrone et al., “Passive Immunization WithAnti-Glucosaminidase Monoclonal Antibodies Protects Mice FromImplant-Associated Osteomyelitis by Mediating Opsonophagocytosis ofStaphylococcus aureus Megaclusters,” J Orthop Res 32(10):1389-96 (2014),which is hereby incorporated by reference in its entirety). Twenty-fourhours later all mice received a trans-tibial pin contaminated withUSA300 LAC::lux, and bioluminescent imaging was performed on Day 3 toconfirm the infection (FIG. 7A). The mice were euthanized 14 days afterinfection, and the tibiae were harvested for micro-CT analysis.Representative 3D renderings of the infected tibiae are shown from themedial and lateral side (FIG. 7B) to illustrate the relative level ofosteolysis in each group (B) of the tibias. The osteolytic volume ineach tibia was quantified using the formula: Osteolytic volume(mm³)=[medial osteolytic area+lateral osteolytic area (mm²)]X corticalthickness (mm) (*p<0.05 vs. PBS). The results are illustratedgraphically in FIG. 7C.

FIGS. 8A-C illustrate the effects of passive immunization with Amd1.6,which show significantly reduced bacterial spread as evidenced by theformation of fewer abscesses in the medullary canal. 6-10 week old,female Balb/c mice (n=5) were immunized intraperitoneally with PBS(negative control), anti-Gmd mAb 1C11, anti-Amd mAb Amd1.6 or acombination (1C11+Amd1.6) at a total dose of 40 mg/kg. Twenty-four hourslater each mouse had inserted through its right tibia a pin contaminatedwith USA300 LAC::lux, a bioluminescent CA-MRSA strain. The resultinginfection was allowed to progress for fourteen days when the animalswere sacrificed and the infected tibiae were harvested, fixed,decalcified and sectioned for histological analysis. Representativeinfected tibiae stained with Orange G/alcian blue (ABG/OH) are depictedfor (FIG. 8A) untreated controls and (FIG. 8B) mice treated with thecombination of anti-Gmd 1C11 and anti-Amd Amd1.6. The number ofabscesses observed in each group of mice is presented in (FIG. 8C). *,p≤0.05; **, p≤0.01.

FIGS. 9A-H illustrate the effect of passive immunization with anti-Amd,anti-Gmd, and a combination of anti-Amd and anti-Gmd monoclonalantibodies in preventing formation of staphylococcal abscess communities(SACs), which leads to sterile abscesses. Mice (n=5) were immunized i.p.with PBS (negative control) or mAbs 1C11, Amd1.6 or a combination(1C11+Amd1.6) at a 40 mg/kg dose. Twenty-four hours later all micereceived a trans-tibial pin contaminated with USA300 LAC::luxbioluminescent CA-MRSA strain. Representative infected tibias from Day14 post-infection are shown for histology sections that wereGram-stained to reveal the bacteria. PBS-treated tibias show typical SACpathology, containing a central nidus of bacteria surrounded by aneosinophilic pseudocapsule within the abscess area (FIGS. 9A-B) that areabsent in mice treated with the following mAbs: 1C11 (FIGS. 9C-D),Amd1.6 (FIGS. 9E-F), and combination 1C11+Amd1.6 (FIGS. 9G-H).

FIGS. 10A-H illustrate the effect of passive immunization with anti-Amd,anti-Gmd, and a combination of anti-Amd and anti-Gmd monoclonalantibodies on recruitment of macrophage-like cells within the abscess.Six-to-ten week old female Balb/c mice (n=5) were immunized i.p. withPBS or mAb 1C11, Amd1.6 or a combination (1C11+Amd1.6) at a 40 mg/kgdose. Twenty-four hours later all mice received an trans-tibial pincontaminated with USA300 LAC::lux bioluminescent CA-MRSA strain.Representative infected tibias from Day 14 post-infection are shown forhistology sections that were stained with Orange G/alcian blue (ABG/OH).Passive immunization with anti-Amd, anti-Gmd, and a combination ofanti-Amd and anti-Gmd mAbs recruits macrophage-like cells to the centerof abscess (FIGS. 10C-H, arrowheads) while the PBS immunized mice do notshow macrophage-like cell recruitment within the abscess (FIGS. 10A-B)and display cells that morphologically resemble neutrophils. Multipleabscesses are present in PBS treated tibias (FIG. 10A) in the medullarycanal and soft tissue around the bone, compared to a single abscess inAmd1.6 and combination 1C11+Amd1.6 treated mice (FIGS. 10E and 10G,respectively), or two abscess structures in 1C11 treated mice (FIG.10C).

FIGS. 11A-E illustrate the effect of passive immunization with acombination of anti-Amd and anti-Gmd monoclonal antibodies, whichaccelerates bone healing by recruiting M2 macrophages within the sterileabscess. Six-to-ten week old female Balb/c mice (n=5) were immunizedi.p. with PBS or mAb 1C11, Amd1.6 or a combination (1C11+Amd1.6) at a 40mg/kg dose. Twenty-four hours later all mice received an trans-tibialpin contaminated with USA300 LAC::lux bioluminescent CA-MRSA strain.Representative tibias from Day 14 post-surgery are shown for histologysections that were stained with Orange G/alcian blue (ABG/OH) (FIGS.11A-C). Remarkable healing is evident in mice immunized with the mAbscomparable to those receiving a sterile pin control (FIGS. 11A-C). Todetermine correlation of healing with macrophage phenotype associatedwith remodeling and wound healing process, immunohistochemistry wasperformed with anti-Arginase-1 antibody to stain M2 macrophages. M2macrophages are recruited to the center of the abscess (brown staining)on mice that were passively immunized (FIG. 11E), but excluded fromabscess center on negative control PBS group (FIG. 11D).

DETAILED DESCRIPTION

Disclosed herein are one or more monoclonal antibodies or bindingportions thereof that binds specifically to a Staphylococcus spp.autolysin N-acetylmuramoyl-L-alanine amidase (Amd) catalytic domain orcell wall binding domain.

As used herein, the term “antibody” is meant to include immunoglobulinsderived from natural sources or from recombinant sources, as well asimmunoreactive portions (i.e. antigen binding portions) ofimmunoglobulins. The monoclonal antibodies disclosed herein may exist inor can be isolated in a variety of forms including, for example,substantially pure monoclonal antibodies, antibody fragments or bindingportions, chimeric antibodies, and humanized antibodies (Ed Harlow andDavid Lane, USING ANTIBODIES: A LABORATORY MANUAL (Cold Spring HarborLaboratory Press, 1999), which is hereby incorporated by reference inits entirety).

The monoclonal antibodies disclosed herein are characterized byspecificity for binding to StaphylococcusN-acetylmuramoyl-L-alanine-amidase or fragments thereof. The antibodyspecifically binds to an epitope, typically though not exclusively animmuno-dominant epitope, in the amidase sub-unit of Staphylococcusautolysin (Atl). In certain embodiments, these monoclonal antibodiesinhibit in vivo growth of a Staphylococcus strain. In other embodiments,these monoclonal antibodies inhibit biofilm establishment on metal,plastic and/or organic surfaces. In still further embodiments, one ormore monoclonal antibodies can be used together to inhibit both in vivogrowth of a Staphylococcus strain and biofilm establishment on metal,plastic and/or organic surfaces.

In accordance with this and all other aspects disclosed herein, theStaphylococcus strain is a strain that is, or can be, pathogenic tohumans or animals. The Staphylococcus can be either coagulase-positiveor coagulase-negative. Exemplary Staphylococcus strains include, withoutlimitation, S. aureus, S. epidermidis, S. lugdunensis, S. saprophyticus,S. haemolyticus, S. caprae, and S. simiae. In one embodiment, themonoclonal antibodies disclosed herein are effective againstantibiotic-resistant strains of Staphylococcus, includingmethicillin-resistant or vancomycin-resistant strains.

In certain embodiments, the epitope of the amidase subunit (that isbound by the mAb or binding fragment thereof) is an immuno-dominantepitope. Immuno-dominant antigen is a part of the antigenic determinantthat is most easily recognized by the immune system and thus exerts themost influence on the specificity of the induced antibody. An“immuno-dominant epitope” refers to the epitope on an antigen thatselectively provokes an immune response in a host organism to thesubstantial exclusion of other epitopes on that antigen.

Usually, the antigen likely to carry an immuno-dominant epitope can beidentified by selecting antigens on the outer surface of the pathogenicorganism. For example, most simple organisms, such as fungi, bacteriaand viruses have one or two proteins that are exposed on the outersurface of the pathogenic organism. These outer surface proteins aremost likely to carry the appropriate antigen. The proteins most likelyto carry an immuno-dominant epitope can be identified in a Western assayin which total protein is run on a gel against serum from an organisminfected with the pathogenic organism. Bound antibodies from the serumare identified by labeled anti-antibodies, such as in one of thewell-known ELISA techniques. The immuno-dominant epitope can beidentified by examining serum from a host organism infected with thepathogenic organism. The serum is evaluated for its content ofantibodies that bind to the identified antigens that are likely to causean immune response in a host organism. If an immuno-dominant epitope ispresent in these antigens, substantially all antibodies in the serumwill bind to the immuno-dominant epitope, with little binding to otherepitopes present in the antigen.

AtlA is one of the catalytically distinct peptidoglycan hydrolases inStaphylococcus aureus that is required to digest the cell wall duringmitosis (Baba and Schneewind, “Targeting of Muralytic Enzymes to theCell Division Site of Gram-Positive Bacteria: Repeat Domains DirectAutolysin to the Equatorial Surface Ring of Staphylococcus aureus,”EMBO. J. 17(16):4639-46 (1998), which is hereby incorporated byreference in its entirety). In addition to being an essential gene forgrowth, scanning electron microscopy studies have demonstrated thatanti-AtlA antibodies bound to S. aureus during binary fission localizeto regions of the bacteria that are not covered by the cell wall (Yamadaet al., “An Autolysin Ring Associated With Cell Separation ofStaphylococcus aureus,” J. Bacteriol. 178(6):1565-71 (1996), which ishereby incorporated by reference in its entirety).

The AtlA enzyme is comprised of an amidase (62 kD) and glucosaminidase(53 kD), which are produced from the same AtlA precursor protein via acleavage process (Baba and Schneewind, “Targeting of Muralytic Enzymesto the Cell Division Site of Gram-Positive Bacteria: Repeat DomainsDirect Autolysin to the Equatorial Surface Ring of Staphylococcusaureus,” Embo. J. 17(16):4639-46 (1998); Komatsuzawa et al.,“Subcellular Localization of the Major Autolysin, ATL and Its ProcessedProteins in Staphylococcus aureus,” Microbiol Immunol. 41:469-79 (1997);Oshida et al., “A Staphylococcus aureus Autolysin That Has anN-acetylmuramoyl-L-alanine Amidase Domain and anEndo-beta-N-acetylglucosaminidase Domain: Cloning, Sequence Analysis,and Characterization,” Proc. Nat'l. Acad. Sci. U.S.A. 92(1):285-9(1995), which are hereby incorporated by reference in their entirety).The autolysin is held to the cell wall by three ˜150 amino acid cellwall binding domains, which are designated as R1, R2, and R3. In thefinal maturation step, proteolytic cleavage separates the amidase domainand its associated R1 and R2 domains (collectively, “Amd”) from theglucosaminidase and its associated N-terminal R3 domain (collectively,“Gmd”). See FIG. 1.

Exemplary encoded consensus protein and encoding open reading framesequences for His-Amd are identified as SEQ ID NOS: 1 and 2 below.

SEQ ID NO: 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 ID NO: 2ATGCACCATCACCACCACCACAGCGCAAGCGCACAGCCTCGTTCCGTCGCCGCCACCCCGAAAACCAGCTTGCCGAAGTACAAACCGCAAGTTAATAGCAGCATCAACGACTACATCCGCAAAAACAACCTGAAGGCCCCGAAAATTGAAGAGGACTATACCAGCTATTTCCCGAAATATGCTTACCGTAATGGTGTCGGTCGTCCGGAGGGTATTGTGGTCCACGACACCGCGAATGACCGTAGCACCATCAACGGTGAGATTAGCTACATGAAAAACAATTACCAAAACGCGTTCGTGCACGCCTTCGTCGATGGCGATCGCATCATCGAAACCGCGCCAACCGACTATCTGTCCTGGGGTGTGGGTGCCGTTGGCAACCCGCGTTTCATCAATGTGGAGATTGTTCATACCCACGACTACGCGAGCTTTGCACGTAGCATGAACAACTACGCCGATTATGCTGCAACGCAGCTGCAGTACTACGGCCTGAAACCGGATAGCGCGGAGTATGACGGTAACGGTACGGTGTGGACGCATTATGCGGTGAGCAAATACCTGGGTGGTACCGATCATGCTGATCCGCATGGCTACCTGCGCTCTCACAACTATAGCTACGACCAGTTGTACGACCTGATCAATGAGAAATATCTGATTAAGATGGGTAAGGTTGCACCGTGGGGTACGCAGAGCACCACGACGCCGACCACGCCGAGCAAACCGACGACCCCGTCCAAACCGTCTACCGGCAAACTGACGGTCGCGGCTAATAACGGTGTCGCGCAGATTAAACCGACCAACAGCGGTCTGTACACCACCGTCTATGATAAAACGGGCAAAGCCACCAATGAGGTTCAAAAGACGTTCGCAGTTAGCAAAACGGCGACCCTGGGTAACCAAAAGTTCTACCTGGTTCAGGATTACAATAGCGGCAACAAATTTGGTTGGGTGAAAGAAGGCGACGTTGTGTACAATACCGCGAAGTCCCCGGTGAACGTTAATCAGAGCTATAGCATCAAGCCGGGTACCAAATTGTATACGGTGCCGTGGGGTACCAGCAAGCAAGTTGCGGGTAGCGTCAGCGGCTCTGGTAACCAGACCTTCAAGGCGTCTAAGCAACAACAAATTGACAAAAGCATTTACCTGTATGGTAGCGTTAATGGTAAAAGCGGCTGGGTGTCTAAAGCGTATCTGGTCGACACCGCAAAGCCGACGCCAACGCCGACCCCGAAGCCGAGCACCCCAACCACCAACAACAAGCTGACGGTCAGCTCCCTGAATGGTGTTGCGCAAATCAATGCGAAGAATAATGGCCTGTTTACCACCGTTTACGATAAGACGGGCAAGCCAACGAAAGAAGTCCAGAAAACCTTTGCTGTCACCAAAGAAGCCAGCCTGGGCGGTAACAAGTTCTATCTGGTTAAGGACTACAACTCCCCGACGCTGATCGGTTGGGTCAAACAAGGCGATGTCATTTACAATAACGCGAAAAGCCCGGTTAATGTGATGCAAACCTATACCGTCAAACCGGGTACGAAGCTGTATTCCGTTCCGTGGGGCACGTACAAACAAGAAGCAGGCGCGGTGAGCGGTACCGGCAATCAGACCTTTAAGGCCACCAAGCAGCAGCAGATCGATAAATCTATTTACTTGTTTGGCACCGTGAATGGCAAGAGCGGTTGGGTTTCTAAGGCATACCTGGCGGTGCCGGCAGCACCGAAGAAGGCGGTGGCGCAGCCAAAGACCGCAG TGAAG

The Staphylococcus Amd can be synthesized by solid phase or solutionphase peptide synthesis, recombinant expression, or can be obtained fromnatural sources. Automatic peptide synthesizers are commerciallyavailable from numerous suppliers, such as Applied Biosystems, FosterCity, Calif. Standard techniques of chemical peptide synthesis are wellknown in the art (see e.g., SYNTHETIC PEPTIDES: A USERS GUIDE 93-210(Gregory A. Grant ed., 1992), which is hereby incorporated by referencein its entirety). Protein or peptide production via recombinantexpression can be carried out using bacteria, such as E. coli, yeast,insect or mammalian cells and expression systems. Procedures forrecombinant protein/peptide expression are well known in the art and aredescribed by Sambrook et al, Molecular Cloning: A Laboratory Manual(C.S.H.P. Press, NY 2d ed., 1989).

Recombinantly expressed peptides can be purified using any one ofseveral methods readily known in the art, including ion exchangechromatography, hydrophobic interaction chromatography, affinitychromatography, gel filtration, and reverse phase chromatography. Thepeptide is preferably produced in purified form (preferably at leastabout 80% or 85% pure, more preferably at least about 90% or 95% pure)by conventional techniques. Depending on whether the recombinant hostcell is made to secrete the peptide into growth medium (see U.S. Pat.No. 6,596,509 to Bauer et al., which is hereby incorporated by referencein its entirety), the peptide can be isolated and purified bycentrifugation (to separate cellular components from supernatantcontaining the secreted peptide) followed by sequential ammonium sulfateprecipitation of the supernatant. The fraction containing the peptide issubjected to gel filtration in an appropriately sized dextran orpolyacrylamide column to separate the peptides from other proteins. Ifnecessary, the peptide fraction may be further purified by HPLC and/ordialysis.

In certain embodiments, the monoclonal antibodies or binding portionsmay bind specifically to an epitope of the Amd catalytic domain. As usedherein, the Amd catalytic domain is at least 70% identical to aminoacids 9-252 of SEQ ID NO: 1, or at least 75% or 80% identical to aminoacids 9-252 of SEQ ID NO: 1, or even at least 85% or 90% identical toamino acids 9-252 of SEQ ID NO: 1. In certain embodiments, the amidasecatalytic domain is at least 95% identical to amino acids 9-252 of SEQID NO: 1.

In certain embodiments, the monoclonal antibody or binding portion isproduced by a hybridoma cell line designated as Amd1.6, Amd1.10,Amd1.13, Amd1.16, Amd1.17, Amd2.1, or Amd2.2.

In another embodiment, the monoclonal antibody or binding portion bindsto an epitope wholly or partly within the Amd R1 or R2 cell wall bindingdomain. As used herein, the R1 or R2 cell wall binding domains are atleast 70% identical to amino acids 253-399 or 421-568 of SEQ ID NO: 1,respectively; or at least 75% or 80% identical to amino acids 253-399 or421-568 of SEQ ID NO: 1, respectively; or even at least 85% or 90%identical to amino acids 253-399 or 421-568 of SEQ ID NO: 1,respectively. In certain embodiments, the cell wall binding domains areat least 95% identical to amino acids 253-399 or 421-568 of SEQ ID NO:1, respectively.

In certain embodiments, the monoclonal antibody or binding portion isproduced by a hybridoma cell line designated Amd1.1, Amd1.2, Amd1.5,Amd1.7, Amd1.8, Amd1.9, Amd1.11, Amd1.12, Amd1.14, Amd1.15, Amd2.4, orAmd2.5.

In certain embodiments the monoclonal antibody disclosed herein binds tothe Amd catalytic domain or cell wall binding domain with an affinitygreater than 10⁻⁸M or 10⁻⁹M, but preferably greater than 10⁻¹⁰ M.

As noted above, in certain embodiments the monoclonal antibodies orbinding portions also inhibit in vivo growth of Staphylococcus.Inhibition of in vivo growth of Staphylococcus can be measured accordingto a number of suitable standards. In one such embodiment, the in vivogrowth of Staphylococcus can be assessed according to a bioluminescenceassay. By way of example, bioluminescent S. aureus (Xen 29; ATCC 12600)(Francis et al., “Monitoring Bioluminescent Staphylococcus aureusInfections in Living Mice Using a Novel luxABCDE Construct,” Infect.Immun. 68(6):3594-600 (2000); see also Contag et al., “PhotonicDetection of Bacterial Pathogens in Living Hosts,” Mol. Microbiol.18(4):593-603 (1995), each of which is hereby incorporated by referencein its entirety) is used to dose a transtibial implant with 500,000 CFUprior to surgical implant. Five week old female BALB/cJ mice can receivean intraperitoneal injection of saline or 1 mg of purifiedantibody/antibody fragment in 0.25 ml saline 3 days prior to surgery.The mice can be imaged to assess bioluminescence on various days (e.g.,0, 3, 5, 7, 11, and 14) and a comparison of BLI images can be comparedto assess whether the antibody inhibits in vivo growth of S. aureusrelative to the saline control or a control mouse injected with aplacebo antibody.

In another embodiment, the in vivo growth of Staphylococcus can beassessed according to biofilm formation. By way of example, femaleBalb/c mice can be passively immunized intraperitoneally withantibody/antibody fragment or control at a dose of 40 mg/kg, and one daylater each mouse can be infected with a trans-tibial stainless steel pincontaminated with a MRSA strain. On day 14 post-infection the pins canbe removed and examined by scanning electron microscopy (SEM), and thepercentage of a region of interest (e.g., 0.5×2.0 mm face of the flatpin) covered with biofilm can be quantified with NIH software (Image J).

In yet another embodiment, the Osteolytic Volume of infected bone can bemeasured using MicroCT imaging. By way of example, female Balb/c micecan be passively immunized intraperitoneally with antibody/antibodyfragment or control at a dose of 40 mg/kg, and one day later each mousecan be infected with a trans-tibial stainless steel pin contaminatedwith a MRSA strain. After 14 days, the mice can be euthanized and thetibia harvested. Using the resulting images, the lesion area can bemeasured in two different views (e.g., medial and lateral), which areadded together and multiplied by the cortical thickness (see Varrone etal., “Passive Immunization With Anti-Glucosaminidase MonoclonalAntibodies Protects Mice From Implant-Associated Osteomyelitis byMediating Opsonophagocytosis of Staphylococcus aureus Megaclusters,” JOrthop Res 32(10):1389-96 (2014), which is hereby incorporated byreference in its entirety).

In yet another embodiment, in vivo growth of Staphylococcus can beassessed by the presence (including frequency) or absence ofStaphylococcus abscess communities (SACs) in the medullary canal or softtissue surrounding the bone. By way of example, female Balb/c mice canbe passively immunized intraperitoneally with antibody/antibody fragmentor control at a dose of 40 mg/kg, and one day later each mouse can beinfected with a trans-tibial stainless steel pin contaminated with aMRSA strain. After 14 days, the mice can be euthanized and the tibia andassociated soft tissue harvested. Histological samples can be preparedand stained with Orange G/alcian blue (ABG/OH), and then the presence orabsence of abscesses can be determined upon analysis of the histologicsamples.

According to one embodiment, the monoclonal antibody or binding portioncomprises a V_(H) domain comprising one of the following amino acidsequences (CDR domains underlined):

SEQ ID NO: 5 (Amd1.2):PELVKPGASVKMSCKASGYTFTSYIMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSTTAYMELSSLTSEDXAVYYCARLDGYYDCFDYWGQGTTLTVSSwhere X can be any amino acid. This amino acid sequence is encoded by thefollowing nucleotide sequence (SEQ ID NO: 6):CCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGCTATATTATGCACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACAATGATGGTACTAAGTACAATGAGAAGTTCAAAGGCAAGGCCACACTGACTTCAGACAAATCCTCCACCACAGCCTACATGGAGCTCAGCAGCCTGACCTCTGAGGACTNTGCGGTCTATTACTGTGCAAGACTTGATGGTTACTACGACTGCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCNTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCNGGTCAAGGGwhere each N can be A, T, C, or G, as long as the nucleic acid molecule encodesthe amino acid sequence of SEQ ID NO: 5. SEQ ID NO: 7 (Amd1.1):QQSGAELVKPGASVKLSCTASGFNIKDTYIHWVKQRPEQGLEWIGRIDPANGITNYDPKFQGRATITADTSSNIAYLQLTSLTSEGTAVYYCARGGYLSPYAMDYWGQGTSVTVSSThis amino acid sequence is encoded by the following nucleotide sequence (SEQID NO: 8):NTGCAGCAGTCTGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACACCTATATACATTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTATTACTAATTATGACCCGAAGTTCCAGGGCAGGGCCACTATAACAGCAGACACATCCTCCAATATAGCCTACCTGCAGCTCACCAGCCTGACATCTGAGGGCACTGCCGTCTACTACTGTGCTAGAGGGGGTTACCTATCCCCTTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTNCCCTGAGCCAGwhere each N can be A, T, C, or G, as long as the nucleic acid molecule encodesthe amino acid sequence of SEQ ID NO: 7. SEQ ID NO: 9 (Amd1.5):QQSGAELVRPGALVKLSCKASGFNIQDYYLHWMKQRPEQGLEWIGWIDPENDNTVYDPKFRDRASLTADTFSNTAYLQLSGLTSEDTAVYYCARRDGITTATRAMDYWGQGTSTVSSThis amino acid sequence is encoded by the following nucleotide sequence (SEQID NO: 10):TGCAGCAGTCTGGGGCTGAGCTTGTGAGGCCAGGGGCCTTAGTCAAATTGTCCTGCAAAGCTTCTGGCTTCAACATTCAAGACTACTATCTACACTGGATGAAACAGAGGCCTGAGCAGGGCCTGGAGTGGATTGGATGGATTGATCCTGAGAATGATAATACTGTATATGACCCGAAGTTCCGGGACAGGGCCAGTTTAACAGCAGACACATTTTCCAACACAGCCTACCTACAGCTCAGCGGCCTGACATCTGAAGACACTGCCGTCTATTACTGTGCTAGAAGAGACGGCATTACTACGGCTACGCGGGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCNNNNNCCTGAGCCAGwhere each N can be A, T, C, or G, as long as the nucleic acid molecule encodesthe amino acid sequence of SEQ ID NO: 9. SEQ ID NO: 11 (Amd1.6):QSGTVLARPGTSVKMSCKASGYSFTNYWMHWVRQRPGQGLEWIGSIYPGNSDTTYNQKFKDKAKLTAVTSASTAYMELSSLTNEDSAVYYCTGDDYSRFSYWGQGTLVTVSAThis amino acid sequence is encoded by the following nucleotide sequence (SEQID NO: 12):CAGTCTGGGACTGTACTGGCAAGGCCTGGGACTTCCGTGAAGATGTCCTGCAAGGCTTCTGGCTACAGCTTTACCAACTACTGGATGCACTGGGTAAGACAGAGGCCTGGACAGGGTCTAGAATGGATTGGTTCTATTTATCCTGGAAATAGTGATACTACCTACAACCAGAAGTTCAAGGACAAGGCCAAACTGACTGCAGTCACATCCGCCAGCACTGCCTACATGGAGCTCAGCAGCCTGACAAATGAGGACTCTGCGGTCTATTACTGTACGGGGGATGATTACTCTCGGTTTTCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTNGTCAAGGGCTNTTTCCCNGAGCCAwhere each N can be A, T, C, or G, as long as the nucleic acid molecule encodesthe amino acid sequence of SEQ ID NO: 11. SEQ ID NO 13: (Amd1.7):QQSGPELVKPGASVKISCKASGYTFTDYNMHWVKQSHGKSLEWIGYIFPYNGDTDYNQKFKNKATLTVDNSSSTAYMDLRSLTSEDSAVYYCSRWGSYFDYWGQGTTLTVSSThis amino acid sequence is encoded by the following nucleotide sequence (SEQID NO: 14):TGCAGCAGTCAGGACCTGAGCTGGTGAAACCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTTCTGGATACACATTCACTGACTACAACATGCACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGATATATTTTTCCTTACAATGGTGATACTGACTACAACCAGAAATTCAAGAACAAGGCCACATTGACTGTAGACAATTCCTCCAGCACAGCCTACATGGACCTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTTCAAGATGGGGGTCTTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCT GCCCAAACTAACT CCAT GGT GACCCT GGGAT GCCTGNGTCAAGGGCTwhere each N can be A, T, C, or G, as long as the nucleic acid molecule encodesthe amino acid sequence of SEQ ID NO: 13. SEQ ID NO: 15 (Amd1.9):VESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPKKSLEWVASITSGGSAYYPDSVKGRFTISRDNARNILNLQMSSLRSEDTAMYYCARDDGYFDYWGQGTTLTVSSThis amino acid sequence is encoded by the following nucleotide sequence (SEQID NO: 16):GTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCCAGACTCCAAAAAAGAGTCTGGAGTGGGTCGCATCCATTACTAGTGGTGGTAGCGCCTACTATCCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAGGAACATCCTGAACCTGCAGATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGAGACGACGGGTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAA SEQ ID NO: 17 (Amd1.11):QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLEWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLLINNLKNEDTATYFCARRDGYFDAMDYWGQGTSVTVSSThis amino acid sequence is encoded by the following nucleotide sequence (SEQID NO: 18):NNCCTGATGGCAGCTGCCCAAAGTGCCCAAGCACAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAGAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACTTATGCTGATGACTTCAAGGGACGCTTTGCCITCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCTGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAAGAAGGGATGGTTACTTCGATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGwhere each N can be A, T, C, or G, as long as the nucleic acid molecule encodesthe amino acid sequence of SEQ ID NO: 17. SEQ ID NO: 19 (Amd1.12):QQSGAELVRPGTSVKVSCKTSGYAFTNYLIEWVNQRPGQGLEWIGVINPGSGGTNYNEKFKAKATLTADKSSSTAYMQLSSLTSDDSAVYFCARSERGYYGNYGAMDYWGQGTSVTVSSThis amino acid sequence is encoded by the following nucleotide sequence (SEQID NO: 20):NNGCAGCAGTCTGGAGCTGAGCTGGTAAGGCCTGGGACTTCAGTGAAGGTGTCCTGCAAGACTTCTGGATACGCCTTCACTAATTACTTGATAGAGTGGGTAAATCAGAGGCCTGGACAGGGCCTTGAGTGGATTGGGGTGATTAATCCTGGAAGTGGTGGTACTAACTACAATGAGAAGTTCAAGGCCAAGGCAACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCAGCTCAGCAGCCTGACATCTGATGACTCTGCGGTCTATTTCTGTGCAAGATCAGAGCGAGGCTACTATGGTAACTACGGAGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATNTCCCTGAGCCAGwhere each N can be A, T, C, or G, as long as the nucleic acid molecule encodesthe amino acid sequence of SEQ ID NO: 19. SEQ ID NO: 21 (Amd1.13):QQPGPELVKPGASLKISCKASGYSFSSSWMNWVKQRPGQGLEWIGRIYPVDGDTNYNGKFKGKATLITDKSSSTAYMQLSSLTSVDSAVYFCARTGPYAMDYWGRGTSVTVSSThis amino acid sequence is encoded by the following nucleotide sequence (SEQID NO: 22):NNGCAGCAGCCTGGACCTGAGCTGGTGAAGCCTGGGGCCTCACTGAAGATTTCCTGCAAAGCTTCTGGCTACTCATTCAGTTCCTCTTGGATGAACTGGGTGAAGCAGAGGCCTGGACAGGGTCTTGAGTGGATTGGACGGATTTATCCTGTAGATGGAGATACTAACTACAATGGGAAGTTCAAGGGCAAGGCCACACTGACTACAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACCTCTGTGGACTCTGCGGTCTATTTCTGTGCAAGAACTGGGCCCTATGCTATGGACTACTGGGGTCGAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGwhere each N can be A, T, C, or G, as long as the nucleic acid molecule encodesthe amino acid sequence of SEQ ID NO: 21. SEQ ID NO: 23 (Amd1.16):GAELVRPGSSVKISCKASGYTFSTYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCARSMVTNYYFAMDYWGQGTSVTVSSThis amino acid sequence is encoded by the following nucleotide sequence (SEQID NO: 24):GGGGCTGAGCTGGTGAGGCCTGGGTCCTCAGTGAAGATTTCCTGCAAGGCTTCTGGCTATACATTCAGTACCTACTGGATGAACTGGGTGAAGCAGAGACCTGGACAGGGTCTTGAGTGGATTGGACAGATTTATCCTGGAGATGGTGATACTAACTACAATGGAAAATTCAAGGGTAAAGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTAACATCTGACGACTCTGCGGTCTATTTCTGTGCAAGATCGATGGTAACGAACTATTACTTTGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCNGGTCAAGGGwhere each N can be A, T, C, or G, as long as the nucleic acid molecule encodesthe amino acid sequence of SEQ ID NO: 23. SEQ ID NO: 25 (Amd1.17):GGLVKPGGSLKLSCAASGFTFSDYYMYWVRQTPEKKLEWVATISDGGSYTYYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYCVRGLLGFDYWGQGTTLTVSSThis amino acid sequence is encoded by the following nucleotide sequence (SEQID NO: 26):GGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGGTTCGCCAGACTCCGGAAAAGAAACTGGAGTGGGTCGCAACCATTAGTGATGGTGGTAGTTACACCTACTATCCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACAACCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGTAAGGGGGCTACTGGGTTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGG SEQ ID NO: 27 (Amd2.1):GFVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEMRLEWVASISSGSSXTYYPDSVMGRFTISRDNARNILNLQMSSLRSEDTAMYYCARVGLYYDYYYSMDYWGQGTSVTVSSwhere X can be any amino acid. This amino acid sequence is encoded by thefollowing nucleotide sequence (SEQ ID NO: 28):GGCTTCGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCCAGACTCCAGAGATGAGGCTGGAGTGGGTCGCATCCATTAGTAGTGGTGGTAGNNNCACCTACTATCCAGACAGTGTGATGGGCCGATTCACCATCTCCAGAGATAATGCCAGGAACATCCTGAACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGAGTGGGTCTCTACTATGATTATTACTATTCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGwhere each N can be A, T, C, or G, as long as the nucleic acid molecule encodesthe amino acid sequence of SEQ ID NO: 27. SEQ ID NO: 29 (Amd2.2):ESGPELVKPGASVKISCKASGYTFTDYNMHWVRQSHGKSLEWIGYIYPYNGGTGYNQKFKSKATLTVDNSSSTAYMELRSLTSEDSAVYYCAREDGYYGYFDYWGQGTTLTGSSThis amino acid sequence is encoded by the following nucleotide sequence (SEQID NO: 30):GAGTCAGGACCTGAGCTGGTGAAACCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTTCTGGATACACATTCACTGACTATAACATGCACTGGGTGAGGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGATATATTTATCCTTACAATGGTGGTACTGGCTACAACCAGAAGTTCAAGAGTAAGGCCACATTGACTGTAGACAATTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGAGGATGGTTACTACGGCTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGGCTCCTCAG SEQ ID NO: 31 (Amd 2.4):QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASAAYLQINNLKNEDTATYFCARDYDGYYYYAMDYWGQGTSVTVSSThis amino acid sequence is encoded by the following nucleotide sequence (SEQID NO: 32):CAGATCCAGTTGOTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCGCTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAAGGGACTATGATGGTTACTATTACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAG

According to one embodiment, the monoclonal antibody or binding portioncomprises a V_(L) domain comprising one of the following amino acidsequences (CDR domains underlined):

SEQ ID NO: 33 (Amd1.1):ENVLTQSPAIMSASLGEKVTMTCRASSSVNYMFWFQQKSDASPKLWIYYTSNLAPGVPARFSGSGSGNSYSLTISSMEGEDAATYYCQEFTSFPYTFGThis amino acid sequence is encoded by the following nucleotide sequence(SEQ ID NO: 34):NTCAGTGTCTCAGTTGTAATGTCCAGAGGAGAAAATGTGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGAGAAGGTCACCATGACCTGCAGGGCCAGCTCAAGTGTAAATTACATGTTCTGGTTCCAGCAGAAGTCAGATGCCTCCCCCAAATTGTGGATTTATTATACATCCAACCTGGCTCCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGAACTCTTATTCTCTCACAATCAGCAGCATGGAGGGTGAAGATGCTGCCACTTATTACTGCCAGGAGTTTACTAGTTTCCCGTACACGTT CGGAwhere each N can be A, T, C, or G, as long as the nucleic acid moleculeencodes the amino acid sequence of SEQ ID NO: 33.SEQ ID NO: 35 (Amd1.2):DIVLTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPQYTFThis amino acid sequence is encoded by the following nucleotide sequence(SEQ ID NO: 36):TTATGCTTTTTTGGATTTCAGCCTCCAGAGGTGATATTGTGCTAACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGATAGCGTCAGTCTTTCCTGCAGGGCCAGCCAAAGTATTAGCAACAACCTACACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAGTATGCTTCCCAGTCCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACTCTCAGTATCAACAGTGTGGAGACTGAAGATTTTGGAATGTATTTCTGTCAACAGAGTAACAGCTGGCCTCAGTACACGTTCGG SEQ ID NO: 37 (Amd1.6):SIVMTQTPKFLLVSAGDRLTITCKASQSVSNDVAWYQQKPGQSPKLLIYYTSNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYNSPWTFGGGTKThis amino acid sequence is encoded by the following nucleotide sequence(SEQ ID NO: 38):CCAGGTCTTCGTATTTCTACTGCTCTGTGTGTCTGGTGCTCATGGGAGTATTGTGATGACCCAGACTCCCAAATTCCTGCTTGTATCAGCAGGAGACAGGCTTACCATAACCTGCAAGGCCAGTCAGAGTGTGAGTAATGATGTAGCTTGGTACCAACAGAAGCCAGGGCAGTCTCCTAAACTGCTGATATACTATACATCCAATCGCTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCAGCACTGTGCAGGCTGAAGACCTGGCAGTTTATTTCTGTCAGCAGGATTATAACTCTCCGTGGACGTTCGGTGGAGGCACCAAG SEQ ID NO: 39 (Amd1.7):SIVMTQTPKFLLVSAGDRLTITCKASQSVSNDVAWYQQKPGQSPKLLIYYTSNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYNSPWTFGGGTKThis amino acid sequence is encoded by the following nucleotide sequence(SEQ ID NO: 40):TGGTGCTCATGGGAGTATTGTGATGACCCAGACTCCCAAATTCCTGCTTGTATCAGCAGGAGACAGGCTTACCATAACCTGCAAGGCCAGTCAGAGTGTGAGTAATGATGTAGCTTGGTACCAACAGAAGCCAGGGCAGTCTCCTAAACTGCTGATATACTATACATCCAATCGCTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCAGCACTGTGCAGGCTGAAGACCTGGCAGTTTATTTCTGTCAGCAGGATTATAACTCTCCGTGGACGTTCGGTGGAGGCACCA AGCSEQ ID NO: 41 (Amd1.8):DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQRSHESPRLLIKYVSQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPYTFGThis amino acid sequence is encoded by the following nucleotide sequence(SEQ ID NO: 42):CTTGGACTTTTGCTTTTCTGGACTTCAGCCTCCAGATGTGACATTGTGATGACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGATAGAGTCTCTCTTTCCTGCAGGGCCAGCCAGAGTATTAGCGACTACTTACACTGGTATCAACAAAGATCACATGAGTCTCCAAGGCTTCTCATCAAATATGTTTCCCAATCCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATCAGGGTCAGATTTCACTCTCAGTATCAACAGTGTGGAACCTGAAGATGTTGGAGTGTATTATTGTCAAAATGGTCACAGCTTTCCGTACACGTTCGGA SEQ ID NO: 43 (Amd1.9):DIQMTQSPASLSVSVGETVTITCRTSENIFSNFAWYQQQPGKSPQLLVYGATNLADGVPSRFSGSGSGTQYSLKITSLQSEDFGSYYCQHFWGSPWTFThis amino acid sequence is encoded by the following nucleotide sequence(SEQ ID NO: 44):TTACAGATGCCAGATGTGACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGTATCTGTGGGAGAAACTGTCACCATCACATGTCGAACAAGTGAAAATATTTTCAGTAATTTCGCATGGTATCAGCAGCAACCGGGAAAATCTCCTCAGCTCCTGGTCTATGGTGCAACAAACTTAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTATTCCCTCAAGATCACCAGCCTGCAGTCSEQ ID NO: 45 (Amd1.10):QIVLTQSPALMSASPGEKVTMTCSASSSVSYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPYTFGThis amino acid sequence is encoded by the following nucleotide sequence(SEQ ID NO: 46):TCAGTGCCTCAGTCATAATGTCCAGGGGACAAATTGTTCTCACCCAGTCTCCAGCACTCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGTACTGGTACCAGCAGAAGCCAAGATCCTCCCCCAAACCCTGGATTTATCTCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAACCCACCCTACACG TTCGGASEQ ID NO: 47 (Amd1.11):DILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPALTFGThis amino acid sequence is encoded by the following nucleotide sequence(SEQ ID NO: 48):GGACTTTTGCTTTTCTGGATTCCAGCCTCCAGAGGTGACATCTTGCTGACTCAGTCTCCAGCCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAGCATTGGCACAAGCATACACTGGTATCAACAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGAGTCTATCTCTGGGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAACAAAGTAATAGCTGGCCAGCGCTCACGTTCGGT SEQ ID NO: 49 (Amd1.12):DIQMTQSPASLSASVGDTVTITCRASENIYSYLAWYQQKQGKSPQLLVYNAKTFAEGVRSRFSGSGSGTQFSLQITSLQPEDFGSYYCQHHYGSPYTFThis amino acid sequence is encoded by the following nucleotide sequence(SEQ ID NO: 50):TCTGCTGCTGTGGCTTACAGGTGCCAGATGTGACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGATACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTCGCAGAAGGTGTGCGATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGCAGATCACCAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATCATTATGGTTCTCCGTACA CGTTCGGSEQ ID NO: 51 (Amd1.13):DIVMTQSPSSLTVTAGEKVIMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLISWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGThis amino acid sequence is encoded by the following nucleotide sequence(SEQ ID NO: 52):GGTACCTGTGGGGACATTGTGATGACGCAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAAATCAAAAAAACTACTTGACCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTGTTGATCTCCTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAATGACTATAGTTATCCATTCAC GTTCGGCSEQ ID NO: 53 (Amd1.15):DIAMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVRDRFXGSRCGTDFTFPISSVQGEDLAVYYCQQHYSIHSRSwhere X can be any amino acid. This amino acid sequence is encoded by thefollowing nucleotide sequence (SEQ ID NO: 54):NCTGCTATTCTGCTATGGGTATCTGGTGTTGACGGAGACATTGCGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGAGTACTGCTGTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTACTCGGCATCCTACCGGTACACTGGAGTCCGTGATCGCTTCANTGGCAGTCGATGTGGGACGGATTTCACTTTCCCCATCAGCAGTGTGCAGGGTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATAGTATCCATTCACGTTCGGwhere each N can be A, T, C, or G, as long as the nucleic acid moleculeencodes the amino acid sequence of SEQ ID NO: 53.SEQ ID NO: 55 (Amd1.17):DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTThis amino acid sequence is encoded by the following nucleotide sequence(SEQ ID NO: 56):TGGATCCCTGCTTCCAGCAGTGATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAGAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAA SEQ ID NO: 57 (Amd 2.1):DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLYSGNQKNYLTWYQQKPGQPPKMLIYWASTRESGVPDRFTGSGSGTHFTLTISSVAEDLAIYYCQNDYSYPVTFGAGTKLELKThis amino acid sequence is encoded by the following nucleotide sequence(SEQ ID NO: 58):GACATTGTGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTATACAGTGGAAATCAAAAGAACTACTTGACCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAAATGTTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACACATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAATTTATTACTGTCAGAATGATTATAGTTATCCGGTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAC SEQ ID NO: 59 (Amd 2.2):EIVLTQSPAITAASLGQKVTITCSASSSVNYMHWYQQKSGTSPKPWIYEISKLASGVPARFSGSGSGTSYSLTISSMEAEDAAIYYCQQWNYPLITFGAGTKLELKThis amino acid sequence is encoded by the following nucleotide sequence(SEQ ID NO: 60):GAAATTGTGCTCACTCAGTCTCCAGCCATCACAGCTGCATCTCTGGGGCAAAAGGTCACCATCACCTGCAGTGCCAGCTCAAGTGTAAATTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAACCATGGATTTATGAAATATCCAAACTGGCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCATTTATTACTGCCAGCAGTGGAATTATCCTCTTATCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACSEQ ID NO: 61 (Amd 2.4):ENALTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSSMSPKLWIYDTSKLASGVPGRFSGSGSGNSYSLTISSMEAEEVATYYCFQGSGFPVHVRRGDQVGNKTThis amino acid sequence is encoded by the following nucleotide sequence(SEQ ID NO: 62):GAAAATGCTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAAAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGTCAAGCATGTCCCCCAAACTCTGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCAGGTCGCTTCAGTGGCAGTGGGTCTGGAAACTCTTACTCTCTCACGATCAGCAGCATGGAGGCTGAAGAGGTTGCCACTTATTACTGTTTTCAGGGGtAGTGGGTTCCCAGTACACGTTCGGAGGGGGGACCAAGTTGGAAATAAAA CSEQ ID NO: 63 (Amd 2.5):DIQMTQSPASLSASVGETITITCRASGNIHNYLAWYQQKQGKSPHLLVFHARSLADGVPSRFSGSGSGTQYSLNINSLQPEDFGIYYCQHFWYTPYTFGGGTKLEIKThis amino acid sequence is encoded by the following nucleotide sequence(SEQ ID NO: 64):GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTATCACCATCACATGTCGAGCAAGTGGGAATATTCACAATTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCACCTCCTGGTCTTTCATGCAAGATCCTTAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGAACACAATATTCTCTCAATATCAACAGCCTGCAGCCTGAAGATTTTGGGATTTATTACTGTCAACATTTTTGGTATACTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAA AC

Also encompassed by this disclosure are Amd antibodies, and Amd bindingportions thereof, that bind to the same epitope of Amd as one or more ofthe disclosed anti-Amd antibodies. Additional antibodies and Amd bindingantibody portions can therefore be identified based on their ability tocross-compete (e.g., to competitively inhibit the binding of, in astatistically significant manner) with the disclosed antibodies in Amdbinding assays. The ability of a test antibody to inhibit the binding ofan anti-Amd reference antibody disclosed herein to an Amd protein (e.g.,an Amd protein or polypeptide having at least part of the sequence ofSEQ ID NO:1, such as the catalytic domain or amino acids 9-252 of SEQ IDNO: 1 or the cell wall binding domain) demonstrates that the testantibody can compete with the reference antibody for binding to Amd.Such an antibody may, according to non-limiting theory, bind to the sameor a related (e.g., a structurally similar or spatially proximal)epitope on the Amd protein as the reference antibody with which itcompetes. In certain embodiments, the antibody that binds to the sameepitope on Amd as a reference antibody disclosed herein is a humanizedantibody. In certain embodiments, the antibody that binds to the sameepitope on Amd as a reference antibody disclosed herein is a humanantibody. The Amd-binding antibodies and Amd binding antibody portionscan also be other mouse or chimeric Amd-binding antibodies and Amdbinding antibody portions which bind to the same epitope as thereference antibody.

The capacity to block or compete with the reference antibody bindingindicates that an Amd-binding test antibody or Amd-binding antibodyportion binds to the same or similar epitope as that defined by thereference antibody, or to an epitope which is sufficiently proximal tothe epitope bound by the reference Amd-binding antibody. Such antibodiesare especially likely to share the advantageous properties identifiedfor the reference antibody.

The capacity to block or compete with the reference antibody may bedetermined using techniques known in the art such as a competitionbinding assay. With a competition binding assay, the antibody orAmd-binding antibody portion under test is examined for ability toinhibit specific binding of the reference antibody to an Amd protein ora portion of an Amd protein (e.g., the catalytic domain or amino acids9-252 of SEQ ID NO: 1, or the cell wall binding domain). A test antibodycompetes with the reference antibody for specific binding to the Amdprotein or portion thereof, as antigen, if an excess of the testantibody substantially inhibits binding of the reference antibody.Substantial inhibition means that the test antibody reduces specificbinding of the reference antibody usually by at least 10%, 25%, 50%,75%, or 90%.

Known competition binding assays can be generally applied or routinelyadapted to assess competition of an Amd-binding antibody or Amd-bindingantibody portion with the reference Amd-binding antibody for binding toan Amd protein or portion thereof. Such competition binding assaysinclude, but are not limited to solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (ETA), sandwich competition assay (see Stähli et al.,“Distinction of Epitopes by Monoclonal Antibodies,” Methods inEnzymology 92:242-253, (1983), which is hereby incorporated by referencein its entirety); solid phase direct biotin-avidin EIA (see Kirkland etal., “Analysis of the Fine Specificity and Cross-reactivity ofMonoclonal Anti-lipid A Antibodies,” J. Immunol. 137:3614-3619 (1986),which is hereby incorporated by reference in its entirety); solid phasedirect labeled assay, solid phase direct labeled sandwich assay (EdHarlow and David Lane, USING ANTIBODIES: A LABORATORY MANUAL (ColdSpring Harbor Laboratory Press, 1999), which is hereby incorporated byreference in its entirety); solid phase direct label RIA using 1-125label (see Morel et al., “Monoclonal Antibodies to Bovine Serum Albumin:Affinity and Specificity Determinations,” Molec. Immunol. 25:7-15(1988), which is hereby incorporated by reference in its entirety);solid phase direct biotin-avidin ETA (Cheung et al., “Epitope-SpecificAntibody Response to the Surface. Antigen of Duck Hepatitis B Virus inInfected Ducks,” Virology 176:546-552 (1990), which is herebyincorporated by reference in its entirety); and direct labeled RIA(Moldenhauer et al., “Identity of HML-1 Antigen on IntestinalIntraepithelial T Cells and of B-ly7 Antigen on Hairy Cell Leukaemia,”Sand J. Immunol. 32:77-82 (1990), which is hereby incorporated byreference in its entirety). Typically, such an assay involves the use ofpurified antigen bound to a solid surface or cells bearing either ofthese, an unlabeled test Amd-binding antibody and a labeled referenceantibody. Competitive inhibition is measured by determining the amountof label bound to the solid surface or cells in the presence of the testantibody. Usually the test antibody is present in excess. Antibodies andantigen binding antibody portions identified by competition assay(competing antibodies) include antibodies and antigen binding antibodyportions that bind to the same epitope as the reference antibody andantibodies binding to an adjacent epitope sufficiently proximal to theepitope bound by the reference antibody for steric hindrance to occur.

In some embodiments, the antibody, or Amd binding portion thereof, bindsspecifically to Amd and cross competes with an anti-Amd antibodydescribed herein. In further embodiments, the antibody, or Amd bindingportion thereof, binds specifically to Amd and cross competes with ananti-Amd antibody selected from Amd1.1, Amd1.2, Amd1.5, Amd1.6, Amd1.7,Amd1.8, Amd1.9, Amd1.10, Amd1.11, Amd1.12, Amd1.13, Amd1.14, Amd1.15,Amd1.16, Amd1.17, Amd2.1, Amd2.2, Amd2.4, and Amd2.5. In particularembodiments, the antibody, or Amd binding portion thereof, bindsspecifically to Amd and cross competes with antibody Amd1.6. In otherembodiments, the antibody, or Amd binding portion thereof, bindsspecifically to Amd and cross competes with antibody Amd2.1. In furtherembodiments, the antibody, or Amd binding portion thereof, bindsspecifically to Amd, cross competes with one or more of the aboveanti-Amd antibodies, and inhibits Amd catalytic activity.

In additional embodiments, the antibody, or Amd binding portion thereof,binds to the same epitope as an antibody described herein. In furtherembodiments, the antibody, or Amd binding portion thereof, binds to thesame epitope as an antibody selected from Amd1.1, Amd1.2, Amd1.5,Amd1.6, Amd1.7, Amd1.8, Amd1.9, Amd1.10, Amd1.11, Amd1.12, Amd1.13,Amd1.14, Amd1.15, Amd1.16, Amd1.17, Amd2.1, Amd2.2, Amd2.4, and Amd2.5.In particular embodiments, the antibody, or Amd binding portion thereof,binds to the same epitope as antibody Amd1.6. In other embodiments, theantibody, or Amd binding portion thereof, binds to the same epitope asantibody Amd2.1. In further embodiments, the antibody, or Amd bindingportion thereof, binds to the same epitope as one or more of the aboveanti-Amd antibodies and inhibits Amd catalytic activity.

In some embodiments, the antibody, or Amd binding portion thereof, bindsspecifically to Amd and cross competes with an anti-Amd antibody thatbinds a Staphylococcus spp. Amd catalytic domain. In furtherembodiments, the antibody, or Amd binding portion thereof, bindsspecifically to Amd and cross competes with an anti-Amd antibodyselected from Amd1.6, Amd1.10, Amd1.13, Amd1.16, Amd1.17, Amd2.1, andAmd2.2. In further embodiments, the antibody, or Amd binding portionthereof, binds specifically to Amd, cross competes with one or more ofthe above-identified anti-Amd antibodies, and inhibits Amd catalyticactivity.

In additional embodiments, the antibody, or Amd binding portion thereof,binds to the same epitope of an Amd catalytic domain as an antibodydescribed herein. In further embodiments, the antibody, or Amd bindingportion thereof, binds to the same epitope of an Amd catalytic domain asan antibody selected from Amd1.6, Amd1.10, Amd1.13, Amd1.16, Amd1.17,Amd2.1, and Amd2.2. In further embodiments, the antibody, or Amd bindingportion thereof, binds to the same epitope as one or more of theabove-identified anti-Amd antibodies and inhibits Amd catalyticactivity.

In some embodiments, the antibody, or Amd binding portion thereof, bindsspecifically to Amd and cross competes with an anti-Amd antibody thatbinds a Staphylococcus spp. Amd cell wall binding domain. In additionalembodiments, the antibody, or Amd binding portion thereof, bindsspecifically to Amd and cross competes with an anti-Amd antibodydescribed herein that binds a cell wall binding domain. In furtherembodiments, the antibody, or Amd binding portion thereof, bindsspecifically to Amd and cross competes with an antibody selected fromAmd1.1, Amd1.2, Amd1.5, Amd1.7, Amd1.8, Amd1.9, Amd1.11, Amd1.12,Amd1.14, Amd1.15, Amd2.4, and Amd2.5.

In some embodiments, the antibody, or Amd binding portion thereof, bindsto the same epitope of an Amd cell wall binding domain as an anti-Amdantibody described herein. In further embodiments, the antibody, or Amdbinding portion thereof, binds to the same epitope of an Amd cell wallbinding domain as an antibody selected from Amd1.1, Amd1.2, Amd1.5,Amd1.7, Amd1.8, Amd1.9, Amd1.11, Amd1.12, Amd1.14, Amd1.15, Amd2.4, andAmd2.5.

Antibodies disclosed herein may also be synthetic antibodies. Asynthetic antibody is an antibody which is generated using recombinantDNA technology, such as, for example, an antibody expressed by abacteriophage. Alternatively, the synthetic antibody is generated by thesynthesis of a DNA molecule encoding the antibody, followed by theexpression of the antibody (i.e., synthesis of the amino acid specifyingthe antibody) where the DNA or amino acid sequence has been obtainedusing synthetic DNA or amino acid sequence technology which is availableand well known in the art.

In certain embodiments, the synthetic antibody is generated using one ormore of the CDRs of a heavy chain variable domain as identified above,combinations of CDRs from different heavy chain variable domains asidentified above, one or more of the CDRs of a light chain variabledomain as identified, or combinations of CDRs from different light chainvariable domains as identified above. By way of example, Amd1.6 andAmd2.1 include the following CDRs:

Source & CDR Sequence SEQ ID NO: Amd1.6 V_(H), CDR1 GYSFTNYW 65Amd1.6 V_(H), CDR2 IYPGNSDT 66 Amd1.6 V_(H), CDR3 DDYSRFSY 67Amd1.6 V_(L), CDR1 QSVSND 68 Amd1.6 V_(L), CDR2 YTS 69Amd2.1 V_(H), CDR1 GFTFSSYA 70 Amd2.1 V_(H), CDR2 ISSGGSXT 71Amd2.1 V_(H), CDR3 VGLYYDYYYSMDY 72 Amd2.1 V_(L), CDR1 QSLLYSGNQKNY 73Amd2.1 V_(L), CDR2 WAS 74In Amd2.1 V_(H), CDR2 (SEQ ID NO: 71), X can be any amino acid.

In one embodiment, the monoclonal antibody or binding portion ispartially humanized or fully human.

Humanized antibodies are antibodies that contain minimal sequences fromnon-human (e.g. murine) antibodies within the variable regions. Suchantibodies are used therapeutically to reduce antigenicity and humananti-mouse antibody responses when administered to a human subject. Inpractice, humanized antibodies are typically human antibodies withminimum to no non-human sequences. A human antibody is an antibodyproduced by a human or an antibody having an amino acid sequencecorresponding to an antibody produced by a human.

An antibody can be humanized by substituting the complementaritydetermining region (CDR) of a human antibody with that of a non-humanantibody (e.g. mouse, rat, rabbit, hamster, etc.) having the desiredspecificity, affinity, and capability (Jones et al., “Replacing theComplementarity-Determining Regions in a Human Antibody With Those Froma Mouse,” Nature 321:522-525 (1986); Riechmann et al., “Reshaping HumanAntibodies for Therapy,” Nature 332:323-327 (1988); Verhoeyen et al.,“Reshaping Human Antibodies: Grafting an Antilysozyme Activity,” Science239:1534-1536 (1988), which are hereby incorporated by reference intheir entirety). The humanized antibody can be further modified by thesubstitution of additional residues either in the Fv framework regionand/or within the replaced non-human residues to refine and optimizeantibody specificity, affinity, and/or capability.

Humanized antibodies can be produced using various techniques known inthe art. Immortalized human B lymphocytes immunized in vitro or isolatedfrom an immunized individual that produce an antibody directed against atarget antigen can be generated (see e.g. Reisfeld et al., MONOCLONALANTIBODIES AND CANCER THERAPY 77 (Alan R. Liss ed., 1985) and U.S. Pat.No. 5,750,373 to Garrard, which are hereby incorporated by reference intheir entirety). Also, the humanized antibody can be selected from aphage library, where that phage library expresses human antibodies(Vaughan et al., “Human Antibodies with Sub-Nanomolar AffinitiesIsolated from a Large Non-immunized Phage Display Library,” NatureBiotechnology, 14:309-314 (1996); Sheets et al., “Efficient Constructionof a Large Nonimmune Phage Antibody Library: The Production ofHigh-Affinity Human Single-Chain Antibodies to Protein Antigens,” Proc.Nat'l. Acad. Sci. U.S.A. 95:6157-6162 (1998); Hoogenboom et al.,“By-passing Immunisation. Human Antibodies from Synthetic Repertoires ofGermline VH Gene Segments Rearranged in vitro,” J. Mol. Biol. 227:381-8(1992); Marks et al., “By-passing Immunization. Human Antibodies fromV-gene Libraries Displayed on Phage,” J. Mol. Biol. 222:581-97 (1991),which are hereby incorporated by reference in their entirety). Humanizedantibodies can also be made in transgenic mice containing humanimmunoglobulin loci that are capable upon immunization of producing thefull repertoire of human antibodies in the absence of endogenousimmunoglobulin production. This approach is described in U.S. Pat. No.5,545,807 to Surani et al.; U.S. Pat. No. 5,545,806 to Lonberg et al.;U.S. Pat. No. 5,569,825 to Lonberg et al.; U.S. Pat. No. 5,625,126 toLonberg et al.; U.S. Pat. No. 5,633,425 to Lonberg et al.; and U.S. Pat.No. 5,661,016 to Lonberg et al., which are hereby incorporated byreference in their entirety.

In certain embodiments, the humanized monoclonal antibody is IgG1, IgG2,IgG3 class or IgG4 class. The IgG3 class is particularly preferredbecause of its diminished Protein A binding (see Natsume et al.,“Engineered Antibodies of IgG1/IgG3 Mixed Isotype with EnhancedCytotoxic Activities,” Cancer Res 68(10):3863-72 (2008), which is herebyincorporated by reference in its entirety).

Circulating half-life of these antibody classes can be enhanced withmodifications to the Fc domains, such as the N434A and T307A/E380A/N434Asubstitutions described by Petkova et al. (“Enhanced Half-life ofGenetically Engineered Human IgG1 Antibodies in a Humanized FcRn MouseModel: Potential Application in Humorally Mediated Autoimmune Disease,”International Immunology 18(12):1759-1769 (2006), which is herebyincorporated by reference in its entirety) or the N297Q substitutiondescribed by Balsitis et al. (“Lethal Antibody Enhancement of DengueDisease in Mice Is Prevented by Fc Modification,” PloS Pathogens 6(2):e1000790 (2010), which is hereby incorporated by reference in itsentirety).

The heavy and light chain sequences identified above as SEQ ID NOS: 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, and 63, respectively, can beused to identify codon-optimized DNA sequences, which can be introducedinto suitable expression systems for the production of recombinant,chimeric antibodies in accordance with the present invention.Alternatively, the DNA sequences identified above can be used for thepreparation of suitable expression systems for the production ofrecombinant, chimeric antibodies in accordance with the presentinvention.

In addition to whole antibodies, the present invention encompasses Amdbinding portions of such antibodies. Such Amd binding portions include,without limitation, the monovalent Fab fragments, Fv fragments (e.g.,single-chain antibody, scFv), single variable V_(H) and V_(L) domains,and the bivalent F(ab′)₂ fragments, Bis-scFv, diabodies, triabodies, andminibodies. These antibody fragments can be made by conventionalprocedures, such as proteolytic fragmentation procedures, as describedin James Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE 98-118(Academic Press, 1983); Ed Harlow and David Lane, ANTIBODIES: ALABORATORY MANUAL (Cold Spring Harbor Laboratory, 1988); Houston et al.,“Protein Engineering of Antibody Binding Sites: Recovery of SpecificActivity in an Anti-Digoxin Single-Chain Fv Analogue Produced inEscherichia coli,” Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); Birdet al, “Single-Chain Antigen-Binding Proteins,” Science 242:423-426(1988), which are hereby incorporated by reference in their entirety, orother methods known in the art.

In some embodiments, the antibody, or Amd binding portion thereof,comprises a framework in which amino acids have been substituted intothe antibody framework from the respective human V_(H) or V_(L) germlinesequences. Example 6, infra, identifies germline sequences for a numberof antibodies described herein.

It may further be desirable, especially in the case of antibodyfragments, to modify the antibody to increase its serum half-life. Thiscan be achieved, for example, by incorporation of a salvage receptorbinding epitope into the antibody fragment by mutation of theappropriate region in the antibody fragment or by incorporating theepitope binding site into a peptide tag that is then fused to theantibody fragment at either end or in the middle (e.g., by DNA orpeptide synthesis).

Antibody mimics are also suitable for use in accordance with the presentinvention. A number of antibody mimics are known in the art including,without limitation, those known as adnectins or monobodies, which arederived from the tenth human fibronectin type III domain (¹⁰Fn3) (Koideet al., “The Fibronectin Type III Domain as a Scaffold for Novel BindingProteins,” J. Mol. Biol. 284:1141-1151 (1998); Koide et al., “ProbingProtein Conformational Changes in Living Cells by Using Designer BindingProteins: Application to the Estrogen Receptor,” Proc. Natl. Acad. Sci.USA 99:1253-1258 (2002), each of which is hereby incorporated byreference in its entirety); and those known as affibodies, which arederived from the stable alpha-helical bacterial receptor domain Z ofstaphylococcal protein A (Nord et al., “Binding Proteins Selected fromCombinatorial Libraries of an alpha-helical Bacterial Receptor Domain,”Nature Biotechnol. 15(8):772-777 (1997), which is hereby incorporated byreference in its entirety).

In preparing these antibody mimics the CDRs of the V_(H) and/or V_(L)chains can be spliced or grafted into the variable loop regions of theseantibody mimics. The grafting can involve a deletion of at least twoamino acid residues up to substantially all but one amino acid residueappearing in a particular loop region along with the substitution of theCDR sequence. Insertions can be, for example, an insertion of one CDR atone loop region, optionally a second CDR at a second loop region, andoptionally a third CDR at a third loop region. Any deletions,insertions, and replacements on the polypeptides can be achieved usingrecombinant techniques beginning with a known nucleotide sequence (seeinfra).

Methods for monoclonal antibody production may be achieved using thetechniques described herein or others well-known in the art (MONOCLONALANTIBODIES—PRODUCTION, ENGINEERING AND CLINICAL APPLICATIONS (Mary A.Ritter and Heather M. Ladyman eds., 1995), which is hereby incorporatedby reference in its entirety). Generally, the process involves obtainingimmune cells (lymphocytes) from the spleen of a mammal which has beenpreviously immunized with the antigen of interest (i.e., StaphylococcusN-acetylmuramoyl-L-alanine amidase or peptide fragments thereof).

The antibody-secreting lymphocytes are then fused with myeloma cells ortransformed cells, which are capable of replicating indefinitely in cellculture, thereby producing an immortal, immunoglobulin-secreting cellline. Fusion with mammalian myeloma cells or other fusion partnerscapable of replicating indefinitely in cell culture is achieved bystandard and well-known techniques, for example, by using polyethyleneglycol (PEG) or other fusing agents (Milstein and Kohler, “Derivation ofSpecific Antibody-Producing Tissue Culture and Tumor Lines by CellFusion,” Eur. J. Immunol. 6:511 (1976), which is hereby incorporated byreference in its entirety). The immortal cell line, which is preferablymurine, but may also be derived from cells of other mammalian species,is selected to be deficient in enzymes necessary for the utilization ofcertain nutrients, to be capable of rapid growth, and have good fusioncapability. The resulting fused cells, or hybridomas, are cultured, andthe resulting colonies screened for the production of the desiredmonoclonal antibodies. Colonies producing such antibodies are cloned,and grown either in vivo or in vitro to produce large quantities ofantibody.

Thus, a second aspect of present invention relates to a cell line thatexpresses a monoclonal antibody or binding portion disclosed herein. Inone embodiment the monoclonal antibody disclosed herein is produced by ahybridoma cell line designated Amd1.1, Amd1.2, Amd1.3, Amd1.5, Amd1.6,Amd1.7, Amd1.8, Amd1.9, Amd1.10, Amd1.11, Amd1.12, Amd1.13, Amd1.14,Amd1.15, Amd1.16, Amd1.17, Amd2.1, Amd2.2, Amd2.4, and Amd2.5.

As noted above, monoclonal antibodies can be made using recombinant DNAmethods as described in U.S. Pat. No. 4,816,567 to Cabilly et al., whichis hereby incorporated by reference in its entirety. The polynucleotidesencoding a monoclonal antibody are isolated from mature B-cells orhybridoma cells, for example, by RT-PCR using oligonucleotide primersthat specifically amplify the genes encoding the heavy and light chainsof the antibody. The isolated polynucleotides encoding the heavy andlight chains are then cloned into suitable expression vectors, whichwhen transfected into host cells such as E. coli cells, simian COScells, Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, generate host cells thatexpress and secrete monoclonal antibodies. Also, recombinant monoclonalantibodies or fragments thereof of the desired species can be isolatedfrom phage display libraries (McCafferty et al., “Phage Antibodies:Filamentous Phage Displaying Antibody Variable Domains,” Nature348:552-554 (1990); Clackson et al., “Making Antibody Fragments usingPhage Display Libraries,” Nature 352:624-628 (1991); and Marks et al.,“By-Passing Immunization. Human Antibodies from V-Gene LibrariesDisplayed on Phage,” J. Mol. Biol. 222:581-597 (1991), which are herebyincorporated by reference in their entirety).

Still a further aspect relates to a DNA construct comprising a DNAmolecule that encodes an antibody or binding portion disclosed herein, apromoter-effective DNA molecule operably coupled 5′ of the DNA molecule,and a transcription termination DNA molecule operably coupled 3′ of theDNA molecule. The present invention also encompasses an expressionvector into which the DNA construct is inserted. A synthetic gene forthe polypeptides can be designed such that it includes convenientrestriction sites for ease of mutagenesis and uses specific codons forhigh-level protein expression (Gribskov et al., “The Codon PreferencePlot: Graphic Analysis of Protein Coding Sequences and Prediction ofGene Expression,” Nucl. Acids. Res. 12:539-549 (1984), which is herebyincorporated by reference in its entirety).

The gene may be assembled as follows: first the gene sequence can bedivided into parts with boundaries at designed restriction sites; foreach part, a pair of oligonucleotides that code opposite strands andhave complementary overlaps of about 15 bases can be synthesized; thetwo oligonucleotides can be annealed and single strand regions can befilled in using the Klenow fragment of DNA polymerase; thedouble-stranded oligonucleotide can be cloned into a vector, such as,the pET3a vector (Novagen) using restriction enzyme sites at the terminiof the fragment and its sequence can be confirmed by a DNA sequencer;and these steps can be repeated for each of the parts to obtain thewhole gene. This approach takes more time to assemble a gene than theone-step polymerase chain reaction (PCR) method (Sandhu et al., “DualAsymetric PCR: One-Step Construction of Synthetic Genes,” BioTech.12:14-16 (1992), which is hereby incorporated by reference in itsentirety). Mutations could likely be introduced by the low fidelityreplication by Taq polymerase and would require time-consuminggene-editing. Recombinant DNA manipulations can be performed accordingto SAMBROOK & RUSSELL, MOLECULAR CLONING: A LABORATORY MANUAL (2d ed.1989), which is hereby incorporated by reference in its entirety, unlessotherwise stated. To avoid the introduction of mutations during one-stepPCR, high fidelity/low error polymerases can be employed as is known inthe art.

Desired mutations can be introduced to the polypeptide sequence(s) usingeither cassette mutagenesis, oligonucleotide site-directed mutagenesistechniques (Deng & Nickoloff, “Site-Directed Mutagenesis of Virtuallyany Plasmid by Eliminating a Unique Site,” Anal. Biochem. 200:81-88(1992), which is hereby incorporated by reference in its entirety), orKunkel mutagenesis (Kunkel et al., “Rapid and Efficient Site-SpecificMutagenesis Without Phenotypic Selection,” Proc. Natl. Acad. Sci. USA82:488-492 (1985); Kunkel et al., “Rapid and Efficient Site-SpecificMutagenesis Without Phenotypic Selection,” Methods Enzymol. 154:367-382(1987), which are hereby incorporated by reference in their entirety).

Both cassette mutagenesis and site-directed mutagenesis can be used toprepare specifically desired nucleotide coding sequences. Cassettemutagenesis can be performed using the same protocol for geneconstruction described above and the double-stranded DNA fragment codinga new sequence can be cloned into a suitable expression vector. Manymutations can be made by combining a newly synthesized strand (codingmutations) and an oligonucleotide used for the gene synthesis.Regardless of the approach utilized to introduce mutations into thenucleotide sequence encoding a polypeptide according to the presentinvention, sequencing can be performed to confirm that the designedmutations (and no other mutations) were introduced by mutagenesisreactions.

In contrast, Kunkel mutagenesis can be utilized to randomly produce aplurality of mutated polypeptide coding sequences which can be used toprepare a combinatorial library of polypeptides for screening.Basically, targeted loop regions (or C-terminal or N-terminal tailregions) can be randomized using the NNK codon (N denoting a mixture ofA, T, G, C, and K denoting a mixture of G and T) (Kunkel et al., “Rapidand Efficient Site-Specific Mutagenesis Without Phenotypic Selection,”Methods Enzymol. 154:367-382 (1987), which is hereby incorporated byreference in its entirety).

Regardless of the approach used to prepare the nucleic acid moleculesencoding the antibody or Amd binding portion, the nucleic acid can beincorporated into host cells using conventional recombinant DNAtechnology. Generally, this involves inserting the DNA molecule into anexpression system to which the DNA molecule is heterologous (i.e., notnormally present). The heterologous DNA molecule is inserted into theexpression system or vector in sense orientation and correct readingframe. The vector contains the necessary elements (promoters,suppressers, operators, transcription termination sequences, etc.) forthe transcription and translation of the inserted protein-codingsequences. A recombinant gene or DNA construct can be prepared prior toits insertion into an expression vector. For example, using conventionalrecombinant DNA techniques, a promoter-effective DNA molecule can beoperably coupled 5′ of a DNA molecule encoding the polypeptide and atranscription termination (i.e., polyadenylation sequence) can beoperably coupled 3′ thereof.

In accordance with this aspect, the polynucleotides are inserted into anexpression system or vector to which the molecule is heterologous. Theheterologous nucleic acid molecule is inserted into the expressionsystem or vector in proper sense (5′→3′) orientation relative to thepromoter and any other 5′ regulatory molecules, and correct readingframe. The preparation of the nucleic acid constructs can be carried outusing standard cloning methods well known in the art as described bySAMBROOK & RUSSELL, MOLECULAR CLONING: A LABORATORY MANUAL (Cold SpringsLaboratory Press, 2001), which is hereby incorporated by reference inits entirety. U.S. Pat. No. 4,237,224 to Cohen and Boyer, which ishereby incorporated by reference in its entirety, also describes theproduction of expression systems in the form of recombinant plasmidsusing restriction enzyme cleavage and ligation with DNA ligase.

Suitable expression vectors include those which contain replicon andcontrol sequences that are derived from species compatible with the hostcell. For example, if E. coli is used as a host cell, plasmids such aspUC19, pUC18 or pBR322 may be used. When using insect host cells,appropriate transfer vectors compatible with insect host cells include,pVL1392, pVL1393, pAcGP67 and pAcSecG2T, which incorporate a secretorysignal fused to the desired protein, and pAcGHLT and pAcHLT, whichcontain GST and 6×His tags (BD Biosciences, Franklin Lakes, N.J.). Viralvectors suitable for use in carrying out this aspect include, adenoviralvectors, adeno-associated viral vectors, vaccinia viral vectors,nodaviral vectors, and retroviral vectors. Other suitable expressionvectors are described in SAMBROOK AND RUSSELL, MOLECULAR CLONING: ALABORATORY MANUAL (Cold Springs Laboratory Press, 2001), which is herebyincorporated by reference in its entirety. Many known techniques andprotocols for manipulation of nucleic acids, for example in preparationof nucleic acid constructs, mutagenesis, sequencing, introduction of DNAinto cells and gene expression, and analysis of proteins, are describedin detail in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Fred M. Ausubel etal. eds., 2003), which is hereby incorporated by reference in itsentirety.

Different genetic signals and processing events control many levels ofgene expression (e.g., DNA transcription and messenger RNA (“mRNA”)translation) and subsequently the amount of antibodies or antibodyfragments that are produced and expressed by the host cell.Transcription of DNA is dependent upon the presence of a promoter, whichis a DNA sequence that directs the binding of RNA polymerase, andthereby promotes mRNA synthesis. Promoters vary in their “strength”(i.e., their ability to promote transcription). For the purposes ofexpressing a cloned gene, it is desirable to use strong promoters toobtain a high level of transcription and, hence, expression. Dependingupon the host system utilized, any one of a number of suitable promotersmay be used. For instance, when using E. coli, its bacteriophages, orplasmids, promoters such as the T7 phage promoter, lac promoter, trppromoter, recA promoter, ribosomal RNA promoter, the P_(R) and P_(L)promoters of coliphage lambda and others, including but not limited, tolacUV5, ompF, bla, lpp, and the like, may be used to direct high levelsof transcription of adjacent DNA segments. Additionally, a hybridtrp-lacUV5 (tac) promoter or other E. coli promoters produced byrecombinant DNA or other synthetic DNA techniques may be used to providefor transcription of the inserted gene. When using insect cells,suitable baculovirus promoters include late promoters, such as 39Kprotein promoter or basic protein promoter, and very late promoters,such as the p10 and polyhedron promoters. In some cases it may bedesirable to use transfer vectors containing multiple baculoviralpromoters. Common promoters suitable for directing expression inmammalian cells include, without limitation, SV40, MMTV,metallothionein-1, adenovirus Ela, CMV, immediate early, immunoglobulinheavy chain promoter and enhancer, and RSV-LTR. The promoters can beconstitutive or, alternatively, tissue-specific or inducible. Inaddition, in some circumstances inducible (TetOn) promoters can be used.

Translation of mRNA in prokaryotes depends upon the presence of theproper prokaryotic signals, which differ from those of eukaryotes.Efficient translation of mRNA in prokaryotes requires a ribosome bindingsite called the Shine-Dalgarno (“SD”) sequence on the mRNA. Thissequence is a short nucleotide sequence of mRNA that is located beforethe start codon, usually AUG, which encodes the amino-terminalmethionine of the protein. The SD sequences are complementary to the3′-end of the 16S rRNA (ribosomal RNA) and promote binding of mRNA toribosomes by duplexing with the rRNA to allow correct positioning of theribosome. For a review on maximizing gene expression, see Roberts andLauer, “Maximizing Gene Expression on a Plasmid Using Recombination invitro,” Methods in Enzymology, 68:473-82 (1979), which is herebyincorporated by reference in its entirety.

The present invention also includes a host cell transformed with the DNAconstruct disclosed herein. The host cell can be a prokaryote or aeukaryote. Host cells suitable for expressing the polypeptides disclosedherein include any one of the more commonly available gram negativebacteria. Suitable microorganisms include Pseudomonas aeruginosa,Escherichia coli, Salmonella gastroenteritis (typhimirium), S. typhi, S.enteriditis, Shigella flexneri, S. sonnie, S. dysenteriae, Neisseriagonorrhoeae, N. meningitides, Haemophilus influenzae, H.pleuropneumoniae, Pasteurella haemolytica, P. multilocida, Legionellapneumophila, Treponema pallidum, T. denticola, T. orales, Borreliaburgdorferi, Borrelia spp., Leptospira interrogans, Klebsiellapneumoniae, Proteus vulgaris, P. morganii, P. mirabilis, Rickettsiaprowazeki, R. typhi, R. richettsii, Porphyromonas (Bacteroides)gingivalis, Chlamydia psittaci, C. pneumoniae, C. trachomatis,Campylobacter jejuni, C. intermedis, C. fetus, Helicobacter pylori,Francisella tularenisis, Vibrio cholerae, Vibrio parahaemolyticus,Bordetella pertussis, Burkholderie pseudomallei, Brucella abortus, B.susi, B. melitensis, B. canis, Spirillum minus, Pseudomonas mallei,Aeromonas hydrophila, A. salmonicida, and Yersinia pestis.

In addition to bacteria cells, animal cells, in particular mammalian andinsect cells, yeast cells, fungal cells, plant cells, or algal cells arealso suitable host cells for transfection/transformation of therecombinant expression vector carrying an isolated polynucleotidemolecule of the type disclosed herein. Mammalian cell lines commonlyused in the art include Chinese hamster ovary cells, HeLa cells, babyhamster kidney cells, COS cells, and many others. Suitable insect celllines include those susceptible to baculoviral infection, including Sf9and Sf21 cells.

Methods for transforming/transfecting host cells with expression vectorsare well-known in the art and depend on the host system selected, asdescribed in SAMBROOK & RUSSELL, MOLECULAR CLONING: A LABORATORY MANUAL(Cold Springs Laboratory Press, 2001), which is hereby incorporated byreference in its entirety. For bacterial cells, suitable techniquesinclude calcium chloride transformation, electroporation, andtransfection using bacteriophage. For eukaryotic cells, suitabletechniques include calcium phosphate transfection, DEAE-Dextran,electroporation, liposome-mediated transfection, and transduction usingretrovirus or any other viral vector. For insect cells, the transfervector containing the polynucleotide construct is co-transfected withbaculovirus DNA, such as AcNPV, to facilitate the production of arecombinant virus. Subsequent recombinant viral infection of Sf cellsresults in a high rate of recombinant protein production. Regardless ofthe expression system and host cell used to facilitate proteinproduction, the expressed antibodies, antibody fragments, or antibodymimics can be readily purified using standard purification methods knownin the art and described in PHILIP L. R. BONNER, PROTEIN PURIFICATION(Routledge 2007), which is hereby incorporated by reference in itsentirety.

The polynucleotide(s) encoding a monoclonal antibody can further bemodified using recombinant DNA technology to generate alternativeantibodies. For example, the constant domains of the light and heavychains of a mouse monoclonal antibody can be substituted for thoseregions of a human antibody to generate a humanized (or chimeric)antibody, as discussed above. Alternatively, the constant domains of thelight and heavy chains of a mouse monoclonal antibody can be substitutedfor a non-immunoglobulin polypeptide to generate a fusion antibody. Inother embodiments, the constant regions are truncated or removed togenerate the desired antibody fragment of a monoclonal antibody.Furthermore, site-directed or high-density combinatorial mutagenesis ofthe variable region can be used to optimize specificity and affinity ofa monoclonal antibody.

A further aspect relates to a pharmaceutical composition comprising acarrier and one or more monoclonal antibodies or one or more Amd bindingportions thereof in accordance with the present invention. Thispharmaceutical composition may contain two or more antibodies or bindingfragments where all antibodies or binding fragments recognize the sameepitope. Alternatively, the pharmaceutical composition may contain anantibody or binding fragment mixture where one or more antibodies orbinding fragments recognize one epitope of Staphylococcus Amd and one ormore antibodies or binding fragments recognize a different epitope ofStaphylococcus Amd. For example, the mixture may contain one or moreantibodies that bind specifically to an R1 or R2 domain ofStaphylococcus Amd in combination with any other antibody that binds toAmd, such as an antibody that binds to the catalytic domain of Amd. Thepharmaceutical composition may further contain a pharmaceuticallyacceptable carrier or other pharmaceutically acceptable components asdescribed infra. In a preferred embodiment, the carrier is an aqueoussolution.

A pharmaceutical composition containing the antibodies disclosed hereincan be administered to a subject having or at risk of havingStaphylococcus infection. Various delivery systems are known and can beused to administer the antibodies disclosed herein. Methods ofintroduction include but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The therapeutic agent can be administered, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,and the like) and can be administered together with other biologicallyactive agents, such as chemotherapeutic agents, antibiotic agents, orother immunotherapeutic agents. Administration can be systemic or local,i.e., at a site of Staph infection or directly to a surgical or implantsite.

The pharmaceutical composition may also include a second therapeuticagent to the patient, wherein the second therapeutic agent is anantibiotic agent or immunotherapeutic agent. Exemplary antibiotic agentsinclude, without limitation, vancomycin, tobramycin, cefazolin,erythromycin, clindamycin, rifampin, gentamycin, fusidic acid,minocycline, co-trimoxazole, clindamycin, linezolid,quinupristin-dalfopristin, daptomycin, tigecycline, dalbavancin,telavancin, oritavancin, ceftobiprole, ceftaroline, iclaprim, thecarbapenem CS-023/RO-4908463, and combinations thereof. Exemplaryimmunotherapeutic agents include, without limitation, tefibazumab,BSYX-A110, Aurexis™, and combinations thereof. The above lists ofantibiotic agents and immunotherapeutic agents are intended to benon-limiting examples; thus, other antibiotic agents orimmunotherapeutic agents are also contemplated. Combinations or mixturesof the second therapeutic agent can also be used for these purposes.These agents can be administered contemporaneously or as a singleformulation.

In one embodiment, the immunotherapeutic agent includes a secondmonoclonal antibody or binding portion thereof that binds specificallyto a Staphylococcus glucosaminidase (Gmd) and inhibits in vivo growth ofa Staphylococcus strain. Preferably, the second monoclonal antibody isproduced by a hybridoma cell line designated 1C11, 1E12, 2D11, 3A8, 3H6,or 4A12, a humanized variant thereof, or a binding portion thereof (PCTPublication Nos. WO2011/140114 and WO2013/066876 to Schwarz et al.,which are hereby incorporated by reference in their entirety). Also inaccordance with this aspect, the humanized variant of the secondmonoclonal antibody is preferably IgG1, IgG2, IgG3, or IgG4 class.

In another embodiment, the binding portion of the second monoclonalantibody comprises a Fab fragment, Fv fragment, single-chain antibody, aV_(H) domain, or a V_(L) domain.

The pharmaceutical composition typically includes one or morepharmaceutical carriers (e.g., sterile liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like).Water is a more typical carrier when the pharmaceutical composition isadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical excipients include,for example, starch, glucose, lactose, sucrose, gelatin, malt, rice,flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc,sodium chloride, dried skim milk, glycerol, propylene glycol, water,ethanol, and the like. The composition, if desired, can also containminor amounts of wetting or emulsifying agents, or pH buffering agents.These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulations can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the nucleic acid or protein,typically in purified form, together with a suitable amount of carrierso as to provide the form for proper administration to the patient. Theformulations correspond to the mode of administration.

Effective doses of the compositions for the treatment of theabove-described bacterial infections may vary depending upon manydifferent factors, including mode of administration, target site,physiological state of the patient, other medications administered, andwhether treatment is prophylactic or therapeutic. In prophylacticapplications, a relatively low dosage is administered at relativelyinfrequent intervals over a long period of time. Some patients continueto receive treatment for the rest of their lives. In therapeuticapplications, a relatively high dosage at relatively short intervals issometimes required until progression of the disease is reduced orterminated, and preferably until the patient shows partial or completeamelioration of symptoms of disease. Thereafter, the patient can beadministered a prophylactic regime. For prophylactic treatment againstStaphylococcus bacterial infection, it is intended that thepharmaceutical composition(s) disclosed herein can be administered priorto exposure of an individual to the bacteria and that the resultingimmune response can inhibit or reduce the severity of the bacterialinfection such that the bacteria can be eliminated from the individual.For example, the monoclonal antibody or the pharmaceutical compositioncan be administered prior to, during, and/or immediately following asurgical procedure, such as joint replacement or any surgery involving aprosthetic implant.

For passive immunization with an antibody or binding fragment disclosedherein, the dosage ranges from about 0.0001 to about 100 mg/kg, and moreusually about 0.01 to about 10 mg/kg, of the host body weight. Forexample, dosages can be about 1 mg/kg body weight or about 10 mg/kg bodyweight, or within the range of about 1 to about 10 mg/kg. An exemplarytreatment regime entails administration once per every two weeks or oncea month or once every 3 to 6 months. In some methods, two or moremonoclonal antibodies with different binding specificities areadministered simultaneously, in which case the dosage of each antibodyadministered falls within the ranges indicated. Antibody is usuallyadministered on multiple occasions. Intervals between single dosages canbe daily, weekly, monthly, or yearly. In some methods, dosage isadjusted to achieve a plasma antibody concentration of 1-1000 μg/ml andin some methods 25-300 μg/ml. Alternatively, antibody can beadministered as a sustained release formulation, in which case lessfrequent administration is required. Dosage and frequency vary dependingon the half-life of the antibody in the patient. In general, humanantibodies show the longest half-life, followed by humanized antibodies,chimeric antibodies, and nonhuman antibodies.

A further aspect relates to a method of introducing an orthopedicimplant, tissue graft or medical device into a patient that includesadministering to a patient in need of such an implant an effectiveamount of a monoclonal antibody, binding portion, or pharmaceuticalcomposition disclosed herein, and introducing the orthopedic implant ormedical device into the patient.

As used herein, “introducing” a medical device is defined as introducingor installing the device or graft for the first time, as well asresurfacing or otherwise modifying a previously installed device orgraft, replacing—in whole or in part—a previously installed device orgraft, or otherwise surgically modifying a previously installed deviceor graft.

In one embodiment, the method of introducing an orthopedic implant,medical device or graft includes administering to the patient in need ofthe orthopedic implant, medical device or graft an effective amount of amonoclonal antibody or binding fragment or a pharmaceutical compositioncontaining the same, systemically or directly to the site ofimplantation. Alternatively, or in addition, the orthopedic implant,medical device or graft can be coated or treated with the monoclonalantibody or binding fragment or a pharmaceutical composition containingthe same before, during, or immediately after implantation thereof atthe implant site.

The orthopedic implant can be any type of implant that is susceptible toStaphylococcus infection, such as a joint prosthesis, graft or syntheticimplant. Exemplary joint prostheses includes, without limitation, a kneeprosthesis, hip prosthesis, finger prosthesis, elbow prosthesis,shoulder prosthesis, temperomandibular prosthesis, and ankle prosthesis.Other prosthetics can also be used. Exemplary grafts or syntheticimplants include, without limitation, a vascular graft, a heart valveimplant, an artificial intervertebral disk, meniscal implant, or asynthetic or allograft anterior cruciate ligament, medial collateralligament, lateral collateral ligament, posterior cruciate ligament,Achilles tendon, and rotator cuff. Other grafts or implants can also beused.

The medical device can be any medical device that is susceptible toStaphylococcus infection. Exemplary medical devices include, withoutlimitation, a cardiac pacemaker, cerebrospinal fluid shunt, dialysiscatheter, or prosthetic heart valve.

In accordance with this aspect, a second therapeutic agent may also beadministered to the patient. The second therapeutic agent may be anantibiotic agent or immunotherapeutic agent. Exemplary antibiotic agentsand immunotherapeutic agents are described above.

In one embodiment, the method of introducing an orthopedic implant ormedical device is intended to encompass the process of installing arevision total joint replacement. Where infection, particularlyStaphylococcus sp. infection of an original joint replacement occurs,the only viable treatment is a revision total joint replacement. In thisembodiment, the infected joint prosthesis is first removed and then thepatient is treated for the underlying infection. Treatment of theinfection occurs over an extended period of time (i.e. 6 months), duringwhich time the patient is immobile (or has only limited mobility) andreceives high doses of antibiotics to treat the underlying infection andoptionally one or more monoclonal antibodies or binding portions, orpharmaceutical compositions disclosed herein. Upon treatment of theunderlying infection, the new joint prosthesis is installed. Immediatelyprior (i.e., within the two weeks preceding new joint prosthesisinstallation) and optionally subsequent to installation of the new jointprosthesis, the patient is administered one or more monoclonalantibodies or binding portions, or pharmaceutical compositions disclosedherein. This treatment can be repeated one or more times during thepost-installation period. Antibiotic treatment may be administered incombination with or concurrently with the one or more monoclonalantibodies or binding portions, or pharmaceutical compositions disclosedherein. These treatments are effective to prevent infection orreinfection during the revision total joint replacement.

Another aspect relates to a method of treating or preventing aStaphylococcus infection that involves administering to a patientsusceptible to or having a Staphylococcus infection an effective amountof a monoclonal antibody, a monoclonal antibody binding portion, orpharmaceutical composition disclosed herein, or a combination thereof.

In one embodiment of treating Staphylococcus infection, theadministration of the monoclonal antibody, monoclonal antibody bindingportion, pharmaceutical composition, or combination thereof, isrepeated. The initial and repeated administrations can be concurrentwith or in sequence relative to other therapies and carried outsystemically or carried out directly to a site of the Staphylococcusinfection, or both.

The method of treating Staphylococcus infection can be used to treatStaphylococcus infection at sites which include, without limitation,infection of the skin, muscle, cardiac, respiratory tract,gastrointestinal tract, eye, kidney and urinary tract, and bone or jointinfections.

In one embodiment, this method is carried out to treat osteomyelitis byadministering an effective amount of the monoclonal antibody or bindingfragment thereof or the pharmaceutical composition to a patient having aStaphylococcus bone or joint infection. Administration of these agentsor compositions can be carried out using any of the routes describedsupra; in certain embodiments, administration directly to the site ofthe bone or joint infection can be performed.

In each of the preceding embodiments, a second therapeutic agent mayalso be administered to the patient. The second therapeutic agent may bean antibiotic agent or immunotherapeutic agent. Exemplary antibioticagents and immunotherapeutic agents are described above.

The methods of treatment as disclosed herein can be used to treat anypatient in need, including humans and non-human mammals, however, themethods are particularly useful for immuno-compromised patients of anyage, as well as patients that are older than 50 years of age.

In the preceding embodiments, the preventative or therapeutic methods oftreatment can reduce the rate of infection, the severity of infection,the duration of infection, or any combination thereof. In certainembodiments, the preventative or therapeutic methods of treatment canreduce or altogether eliminate the total number of SRCs or abscesses,and/or increase the number of sterile SRCs or abscesses (assuming SRCsor abscesses are present). In certain embodiments, partial or completehealing of an osteolytic lesion is contemplated, as indicated by areduction in lesion size or volume.

Another aspect relates to a method of determining presence ofStaphylococcus in a sample that involves exposing a sample to amonoclonal antibody or binding portion disclosed herein and detectingwhether an immune complex forms between the monoclonal antibody orbinding portion and Staphylococcus or a Staphylococcus amidase presentin the sample, whereby presence of the immune complex after saidexposing indicates the presence of Staphylococcus in the sample.

The sample can be a blood sample, a serum sample, a plasma sample, amucosa-associated lymphoid tissue (MALT) sample, a cerebrospinal fluidsample, an articular liquid sample, a pleural liquid sample, a salivasample, a urine sample, or a tissue biopsy sample.

Detecting formation of an immune complex can be performed by well knownmethods in the art. In one embodiment, the detecting is carried outusing an immunoassay. The immunoassay method used may be a knownimmunoassay method, and for example, common immunoassay methods such aslatex agglutination methods, turbidimetric methods, radioimmunoassaymethods (for example, RIA and RIMA), enzyme immunoassay methods (forexample, ELISA and EIA), gel diffusion precipitation reaction, flowcytometry, immunoelectrophoresis (for example Western blotting), dotblot methods, immunodiffusion assay, protein A immunoassay, fluorescentimmunoassay (for example, FIA and IFMA), immunochromatography methodsand antibody array methods may be mentioned, with no limitation tothese. These immunoassay methods are themselves known in the field, andcan be easily carried out by a person skilled in the art.

The monoclonal antibody or binding portion can be directly labeled byvarious methods known in the art. The label serves as reagent means fordetermining the extent to which the monoclonal antibody or bindingportion is bound by analyte in the immunoassay. The label can be,without limitation, a radioisotope, enzyme, chromophore, fluorophore,light-absorbing or refracting particle. Preferably, the label is aradiolabel, fluorophore, or chemiluminescent label. It is preferable tolabel the antibody or binding portion as extensively as possible withoutdestroying its immunoreactivity.

EXAMPLES

The examples below are intended to exemplify the practicing the claimedsubject matter, but are by no means intended to limit the scope thereof.

Example 1—Preparation of Antigen

A recombinant form of the entire amidase domain of S. aureus autolysinthat includes a hexa-histidine sequence near its N-terminus (His-Amd)was prepared. The open reading frame for His-Amd was designed bycollecting known sequences of S. aureus autolysin, determining theconsensus protein sequence using Geneious™ software, and then optimizingcodon usage for expression in E. coli. The encoded consensus protein andencoding open reading frame sequences for His-Amd are identified as SEQID NOS: 1 and 2 below.

(Hex-histidine leader sequence plus Autolysin aa 198-775) SEQ ID NO: 1MHHHHHHSASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIVVHDTANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASFARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDLINEKYLIKMGKVAPWGTQSITTPTTPSKPTTPSKPSTGKLTVAANNGVAQIKPTNSGLYTTVYDKTGKATNEVQKTFAVSKTATLGNQKFYLVQDYNSGNKFGWVKEGDVVYNTAKSPVNVNQSYSIKPGTKLYTVPWGTSKQVAGSVSGSGNQTFKASKQQQIDKSIYLYGSVNGKSGWVSKAYLVDTAKPTPTPTPKPSTPTTNNKLTVSSLNGVAQINAKNNGLFTTVYDKTGKPTKEVQKTFAVTKEASLGGNKFYLVKDYNSPTLIGWVKQGDVIYNNAKSPVNVMQTYTVKPGTKLYSVPWGTYKQEAGAVSGTGNQTFKATKQQQIDKSIYLFGTVNGKSGWVSKAYLAVPAAPKKAVAQPKTAVK SEQ ID NO: 2ATGCACCATCACCACCACCACAGCGCAAGCGCACAGCCTCGTTCCGTCGCCGCCACCCCGAAAACCAGCTTGCCGAAGTACAAACCGCAAGTTAATAGCAGCATCAACGACTACATCCGCAAAAACAACCTGAAGGCCCCGAAAATTGAAGAGGACTATACCAGCTATTTCCCGAAATATGCTTACCGTAATGGTGTCGGTCGTCCGGAGGGTATTGTGGTCCACGACACCGCGAATGACCGTAGCACCATCAACGGTGAGATTAGCTACATGAAAAACAATTACCAAAACGCGTTCGTGCACGCCTTCGTCGATGGCGATCGCATCATCGAAACCGCGCCAACCGACTATCTGTCCTGGGGTGTGGGTGCCGTTGGCAACCCGCGTTTCATCAATGTGGAGATTGTTCATACCCACGACTACGCGAGCTTTGCACGTAGCATGAACAACTACGCCGATTATGCTGCAACGCAGCTGCAGTACTACGGCCTGAAACCGGATAGCGCGGAGTATGACGGTAACGGTACGGTGTGGACGCATTATGCGGTGAGCAAATACCTGGGTGGTACCGATCATGCTGATCCGCATGGCTACCTGCGCTCTCACAACTATAGCTACGACCAGTTGTACGACCTGATCAATGAGAAATATCTGATTAAGATGGGTAAGGTTGCACCGTGGGGTACGCAGAGCACCACGACGCCGACCACGCCGAGCAAACCGACGACCCCGTCCAAACCGTCTACCGGCAAACTGACGGTCGCGGCTAATAACGGTGTCGCGCAGATTAAACCGACCAACAGCGGTCTGTACACCACCGTCTATGATAAAACGGGCAAAGCCACCAATGAGGTTCAAAAGACGTTCGCAGTTAGCAAAACGGCGACCCTGGGTAACCAAAAGTTCTACCTGGTTCAGGATTACAATAGCGGCAACAAATTTGGTTGGGTGAAAGAAGGCGACGTTGTGTACAATACCGCGAAGTCCCCGGTGAACGTTAATCAGAGCTATAGCATCAAGCCGGGTACCAAATTGTATACGGTGCCGTGGGGTACCAGCAAGCAAGTTGCGGGTAGCGTCAGCGGCTCTGGTAACCAGACCTTCAAGGCGTCTAAGCAACAACAAATTGACAAAAGCATTTACCTGTATGGTAGCGTTAATGGTAAAAGCGGCTGGGTGTCTAAAGCGTATCTGGTCGACACCGCAAAGCCGACGCCAACGCCGACCCCGAAGCCGAGCACCCCAACCACCAACAACAAGCTGACGGTCAGCTCCCTGAATGGTGTTGCGCAAATCAATGCGAAGAATAATGGCCTGTTTACCACCGTTTACGATAAGACGGGCAAGCCAACGAAAGAAGTCCAGAAAACCTTTGCTGTCACCAAAGAAGCCAGCCTGGGCGGTAACAAGTTCTATCTGGTTAAGGACTACAACTCCCCGACGCTGATCGGTTGGGTCAAACAAGGCGATGTCATTTACAATAACGCGAAAAGCCCGGTTAATGTGATGCAAACCTATACCGTCAAACCGGGTACGAAGCTGTATTCCGTTCCGTGGGGCACGTACAAACAAGAAGCAGGCGCGGTGAGCGGTACCGGCAATCAGACCTTTAAGGCCACCAAGCAGCAGCAGATCGATAAATCTATTTACTTGTTTGGCACCGTGAATGGCAAGAGCGGTTGGGTTTCTAAGGCATACCTGGCGGTGCCGGCAGCACCGAAGAAGGCGGTGGCGCAGCCAAAGACCGCAG TGAAG

The DNA molecule encoding His-Amd was synthesized de novo by DNA2.0(Menlo Park, Calif.), and then inserted into the pJexpress E. coliexpression vector.

His-Amd protein expressed in E. coli was primarily in the form ofinsoluble inclusion bodies which were harvested and solubilized in PBSwith 8M urea. After further purification by metal chelationchromatography on TALON resin, the His-Amd was renatured by an extensiveprocess of dialysis against phosphate buffered saline (PBS) containing 1mM Zn²⁺ and stepwise reductions in the level of urea.

The Amd catalytic domain (His-Amd-cat) was prepared in an identicalmanner except that the portion of the open reading frame encoding the R1and R2 domains was omitted (see FIG. 1). The encoded consensus proteinand encoding open reading frame sequences for His-Amd-cat are identifiedas SEQ ID NOS: 3 and 4 below.

(Hex-histidine leader sequence plus Autolysin aa 198-441) SEQ ID NO: 3MHHHHHHSASAQPRSVAATPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPEGIVVHDTANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWGVGAVGNPRFINVEIVHTHDYASFARSMNNYADYAATQLQYYGLKPDSAEYDGNGTVWTHYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDLINEKYLIKMGKVAPWGTQSITTPTTPSKPTTPSKPSTG K SEQ ID NO: 4ATGCACCATCACCACCACCACAGCGCAAGCGCACAGCCTCGTTCCGTCGCCGCCACCCCGAAAACCAGCTTGCCGAAGTACAAACCGCAAGTTAATAGCAGCATCAACGACTACATCCGCAAAAACAACCTGAAGGCCCCGAAAATTGAAGAGGACTATACCAGCTATTTCCCGAAATATGCTTACCGTAATGGTGTCGGTCGTCCGGAGGGTATTGTGGTCCACGACACCGCGAATGACCGTAGCACCATCAACGGTGAGATTAGCTACATGAAAAACAATTACCAAAACGCGTTCGTGCACGCCTTCGTCGATGGCGATCGCATCATCGAAACCGCGCCAACCGACTATCTGTCCTGGGGTGTGGGTGCCGTTGGCAACCCGCGTTTCATCAATGTGGAGATTGTTCATACCCACGACTACGCGAGCTTTGCACGTAGCATGAACAACTACGCCGATTATGCTGCAACGCAGCTGCAGTACTACGGCCTGAAACCGGATAGCGCGGAGTATGACGGTAACGGTACGGTGTGGACGCATTATGCGGTGAGCAAATACCTGGGTGGTACCGATCATGCTGATCCGCATGGCTACCTGCGCTCTCACAACTATAGCTACGACCAGTTGTACGACCTGATCAATGAGAAATATCTGATTAAGATGGGTAAGGTTGCACCGTGGGGTACGCAGAGCACCACGACGCCGACCACGCCGAGCAAACCGACGACCCCGTCCAAACCGTCTACCGGC AAA

Example 2—Inoculation of Mice and Preparation of Hybridomas

For the initial hybridoma fusion (Fusion #1), six female Balb/c micewere immunized two times with 75 μg of His-AmdR1R2, in the SigmaAdjuvant System (Sigma, Cat. No. S6322) by intraperitoneal injection atseven-week intervals. Two of the mice with the highest titers in ELISAon immobilized His-AmdR1R2 were selected for hybridoma fusion. Eachmouse received a final immunization of 350 μg of His-AmdR1R2, i.p., fourdays prior to sacrifice and hybridoma fusion.

For the second hybridoma fusion (Fusion #2), Balb/c mice were immunizedtwo times: first dose with 120 μg of His-AmdR1R2-B from GenScript (LotNumber 222933505/P20011303) in Sigma Adjuvant System (Sigma, Cat. No.S6322), and a second immunization with 100 μg of His-AmdR1R2-Bconjugated with Keyhole limpet hemocyanin (KLH) (Imject EDC mcKLH SpinKit; Thermo Scientific; Cat #77671) at twelve-week intervals. Two of themice with the highest titers in ELISA on immobilized His-AmdR1R2 wereselected for hybridoma fusion. Each mouse received a final immunizationof 100 μg of His-AmdR1R2, i.p., four days prior to sacrifice andhybridoma fusion.

Hybridomas were prepared from splenocytes by conventional methods.

Example 3—Characterization of Monoclonal Antibodies

New monoclonal antibodies were screened on multiple related proteins todetermine that they recognized native Amd (and not just the recombinantform) and whether their epitope was present on the catalytic (C) or cellwall binding domain (R1, R2 or R3). The proteins used for screening themonoclonal antibodies are identified in Table 1 below.

TABLE 1 Proteins Used for Screening the Monoclonal AntibodiesProtein/Antigen SEQ ID Name NO: Region of Autolysin/Sequence DescriptionHis-AmdR1R2  1 MGHHHHHH-Autolysin aa 198 to 775 His-Amdcat  3MGHHHHHH-Autolysin aa 198 to 441 Native Amd 75, 76, 77Mixture of S. aureus UAMS-1 Δspa proteinsincluding full length autolysin, Amd, and Gmd His-AmdR1R2-B 78MGHHHHHH-Autolysin aa 198 to 775-BirA biotinylation site His-R3Gmd-B 79MGHHHHHH-Autolysin aa 776 to 1276-BirA biotinylation site

Screening assays were carried out by ELISA using the proteins identifiedin Table 1 as capture antigen. ELISA tests were performed using widelypracticed conventions. Specifically, antigens were adsorbed onto thewells of NUNC MAXISORP® microtiter plates. Each antigen was prepared asa solution in phosphate-buffered saline (PBS) at 2 μg/mL and 100 μL wasadded to assigned microtiter wells and antigens were allowed to adsorbfor either 1 hour at RT or overnight at 4° C. Wells were blocked by theaddition of 200 μL of 3% bovine serum albumin (BSA), without removal ofthe coating antigen, and incubated for either 1 hour at RT or overnightat 4° C. Coated and blocked plates were then washed 3× with PBSsupplemented with 0.05% TWEEN® 20 (PBS-T) and either used immediately orstored at 4° C.

Cell-free hybridoma culture supernatants were added to assigned wellsand incubated for 1 hour at RT and then washed six times with PBS-T. Thesecondary antibody, horseradish peroxidase-conjugated goat anti-mouseIgG (Southern Biotechnology) was then added, 100 μL per well at 0.1-0.5μg/mL in PBS-T, and incubated 1 hour at RT. Microtiter plates were againwashed six times with PBS-T and then developed by the addition of 100 μLof either 3,3′,5,5′-Tetramethylbenzidine (TMB) or2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). Theresults of these ELISA are shown in Table 2 below.

TABLE 2 Summary of Successfully Cloned and Characterized anti-Amd mAbsAnti- Heavy Amidase IP with Precipi- Amidase Amd Chain Domain nativetation of Enzyme mAh Class C or R1R2 Amidase S. aureus K_(D) Inhibition1.1 IgG1 R1R2 Yes Yes 2.5 nM No 1.2 IgG1 R1R2 Yes Yes ND No 1.4 IgG1R1R2 Yes Yes ND ND 1.5 IgG1 R1R2 Yes Yes ND No 1.6 IgG1 C Yes Yes 2.1 nMYes 1.7 IgG1 R1R2 Yes Yes ND No 1.8 IgG1 R1R2 Yes Yes 2.6 nM No 1.9 IgG1R1R2 Yes Yes 2.6 nM No 1.10 IgG1 C No ND ND ND 1.11 IgG1 R1R2 Yes Yes3.4 nM No 1.12 IgG1 R1R2 No No ND ND 1.13 IgG1 C Yes No ND ND 1.14 IgG1R1R2 No ND ND ND 1.15 IgG1 R1R2 Yes Yes ND ND 1.16 IgG1 C Yes Yes 2.6 nMNo 1.17 IgG1 C Yes No ND No 2.1 IgG1 C Yes Yes 4.9 nM Yes 2.2 IgG1 C YesYes 1.4 nM No 2.4 IgG1 R1R2 Yes Yes 1.9 nM No 2.5 IgG1 R1R2 Yes Yes 6.3nM No ND = Not Determined.

Example 4—Inhibition of Amd Catalytic Activity In Vitro

An attribute that may contribute to the potency of a therapeuticmonoclonal antibody is its direct inhibition of the activity of anenzyme essential for bacterial growth and survival such as amidase. Someof the anti-Amd mAbs were tested for inhibition of amidase activity bymeasuring the extent to which they inhibited the ability of amidase toclarify a turbid suspension of S. aureus peptidoglycan. Results foreight antibodies from Fusion #1 are presented in FIG. 2. MAb Amd1.6 wasa potent inhibitor of amidase activity while the others were not, withthe possible exception of Amd1.16, which appeared to be a low affinityinhibitor. Results for all of the antibodies are summarized in Table 2.

Example 5—the Majority of Anti-Amd mAbs Precipitate S. aureus

Another attribute likely to be important for the potency of therapeuticmonoclonal antibodies is the recognition of antigenic structures(epitopes) accessible from the outside of the intact bacterial cell. Avisible manifestation of this recognition is the antibody-mediatedclustering of individual bacteria into large aggregates that precipitatefrom suspension yielding a cell-rich pellet and a less turbidsupernatant. Many of the candidate mAbs formed conspicuous precipitatesas depicted in FIG. 3. A summary of the precipitation activity of thecandidate mAbs from fusions 1 and 2 is in Table 2.

Example 6—Uniqueness of Each mAb and Identification of Germ LineAssignments Based on Sequencing

Gene assignments were identified by matching nucleotide sequences foranti-Amd heavy and light chains with the files of known murine V-regionsequences in IgBLAST at the National Center for BiotechnologyInformation. The results of this analysis are presented in Table 3below. Each of the antibodies from Fusion #1 was unique except possiblyfor Amd1.1 and 1.4 which were derived from the same germ-line V_(H) genesegments. Two of the antibodies from Fusion#2 share heavy chain V_(H)and J_(H) gene segments with mAbs isolated in Fusion #1 (mAb Amd2.4 withmAb Amd1.11; mAb Amd 2.2 with mAb Amd1.7). In each case the light chainsare distinct.

TABLE 3 Most Probable Germ Line V_(H), J_(H), V_(L) and J_(L) GeneSegments Germ Germ Germ Germ Hybridoma Line V_(H) Line J_(H) Line V_(L)Line J_(L) Amd 1.1 IGHV14-3 (7) IGHJ4 (0) IGKV4-50 (6) IGKJ2 (0) Amd 1.2IGHV1-14 (4) IGHJ2 (0) IGKV5-43 (0) IGKJ2 (0) Amd 1.4 IGHV14-3 (6) IGHJ4(0) NA NA Amd 1.5 IGHV14-1 (11) IGHJ4 (0) NA NA Amd 1.6 IGHV1-5 (8)IGHJ3 (0) IGKV6-32 (3) IGKJ1 (0) Amd 1.7 IGHV1S29 (7) IGHJ2 (0) IGKV6-32(3) IGKJ1 (0) Amd 1.8 NA NA IGKV5-39 (2) IGKJ2 (0) Amd 1.9 IGHV5S12 (5)IGHJ2 (0) IGKV12-46 (8) IGKJ1 (0) Amd 1.10 NA NA IGKV4-68 (0) IGKJ2 (0)Amd 1.11 IGHV9-3-1 (2) IGHJ4 (0) IGKV5-48 (0) IGKJ5 (0) Amd 1.12IGHV1-54 (3) IGHJ4 (0) IGKV12-44 (6) IGKJ2 (0) Amd 1.13 IGHV1-82 (7)IGHJ4 (0) IGKV8-19 (1) IGKJ4 (0) Amd 1.15 NA NA IGKV6-17 (7) IGKJ4 (0)Amd 1.16 IGHV1-80 (6) IGHJ4 (0) NA NA Amd 1.17 IGHV5-4 (2) IGHJ2 (0)IGKV1-117 (1) IGKJ1 (0) Amd 2.1 IGHV5S12 (5) IGHJ4 (0) IGKV8-19 (4)IGKJ5 (0) Amd 2.2 IGHV1S29 (5) IGHJ2 (0) IGKV4-86 (1) IGKJ5 (0) Amd 2.4IGHV9-3-1 (1) IGHJ4 (0) IGKV4-63 (7) IGKJ2 (0) Amd 2.5 NA NA IGKV12-41(9) IGKJ2 (0) Numbers in parentheses are the number of non-synonymousbase changes observed between the anti-Amd sequence and the putativegerm line precursor. NA = sequencing was unsuccessful.

Example 7—Measurement of the Affinity of Anti-Amd mAbs for S. aureusAmidase

An essential attribute of an antibacterial antibody is high affinity forthe bacterial antigen. The higher the affinity, the lower the doserequired for prophylaxis or therapy. While some therapeutic antibodieshave affinities, expressed as K_(D), in the range of 10 nM (K_(A)=10⁸M⁻¹) it is generally desirable to have antibodies with K_(D)˜1 nM(K_(A)˜10⁹ M⁻¹). The affinity of immobilized anti-Amd mAbs for solubleHis-AmdR1R2-B was measured using surface plasmon resonance technology ona Biacore T-200. Representative data for mAb Amd1.6 is presented in FIG.4. While its average affinity for Amd is about 2.1 nM, FIG. 4illustrates a measured affinity of about 1 nM. Measured affinities forthe other candidate mAbs are listed in Table 2.

Example 8—Anti-Amd mAb Amd1.6 Inhibits In Vitro Biofilm Formation by S.aureus Strain UAMS-1

Amd has been reported to be involved in the formation of biofilms (Boseet al., “Contribution of the Staphylococcus aureus Atl AM and GL MureinHydrolase Activities in Cell Division, Autolysis, and BiofilmFormation,” PLoS One 7:e42244 (2012); Chen et al., “Secreted ProteasesControl Autolysin-mediated Biofilm Growth of Staphylococcus aureus,” JBiol Chem. 288:29440-29452. (2013); Houston et al., “Essential Role forthe Major Autolysin in the Fibronectin-binding Protein-mediatedStaphylococcus aureus Biofilm Phenotype,” Infect Immun. 79:1153-1165(2011), each of which is hereby incorporated by reference in itsentirety). Biofilm formation is a process believed to be central to thepersistence of S. aureus infections in vivo, especially those associatedwith orthopedic implants (Ehrlich and Arciola, “From Koch's Postulatesto Biofilm Theory: The Lesson of Bill Costerton,” Internat'l JArtificial Organs 35:695-699 (2012), which is hereby incorporated byreference in its entirety). To measure the ability of anti-Amd mAb1.6 toinhibit biofilm formation, S. aureus strain UAMS-1 was grown in Calgaryplates (Ceri et al., “The Calgary Biofilm Device: New Technology forRapid Determination of Antibiotic Susceptibilities of BacterialBiofilms,” J Clin Microbiol. 37:1771-1776 (1999), which is herebyincorporated by reference in its entirety), which are specificallydesigned for measuring biofilm formation. Deletion mutants in theautolysin gene (Δatl) and in its Amd (Δamd) and Gmd (Δgmd) subdomainseach formed substantially less biofilm than the WT UAMS-1 (20-35% ofWT). Amd1.6 alone or in combination with the anti-Gmd mAb 1C11 (see PCTPublication Nos. WO2011/140114 to Schwarz et al., which is herebyincorporated by reference in its entirety) reduced biofilm formation bymore than 50% while an isotype-matched mAb of irrelevant specificity hadno effect (FIG. 5). Inhibition of the extracellular Amd by exogenousanti-Amd mAb is nearly as effective as deletion of the autolysin gene.

Example 9—Anti-Amd mAb Amd1.6 Reduces Biofilm Formation in an In VivoModel of Implant-Associated Osteomyelitis

Because implant-associated biofilms are thought to be a major source ofpersistence infection in orthopaedic indications, the ability to reducethe extent of biofilm formation on model implants can be interpreted asa measure of the potential clinical benefit of anti-Amd prophylaxis.Using a murine model of implant-associated osteomyelitis in which modelimplant with a defined region of interest, a 0.5×2.0 mm flat face on theimplant, the area that was covered with biofilm during a 14-dayinfection with S. aureus was measured. The maximum extent of infectionis around 40-50% as observed in FIG. 6A where the mice had been treatedwith an isotype-matched antibody of irrelevant specificity. MAb Amd1.6,alone or in combination with the anti-Gmd mAb 1C11, reduced theformation of biofilm by about 50% relative to control (FIGS. 6B, 6C,6E). This degree of reduction in biofilm formation is comparable to thatresulting from a genetic deficiency in the autolysin gene (ADO (FIGS.6B, 6D, 6E), indicating that in terms of biofilm formation the internalgenetic deletion and interference by the exogenous anti-Amd antibody arefunctionally equivalent.

Example 10—Passive Immunization with Anti-Amd mAb Amd1.6 Reduces theVolume of Bone Lysis Resulting from the S. aureus Infection

One of the characteristic features of S. aureus infections in bone isthe lysis of bone resulting from the inflammatory response elicited bythe infecting bacteria. Consequently, reduction in the volume of bonethat is lysed (the Osteolytic Volume) is taken as a measure oflimitation of the infection. To learn if anti-Amd mAb Amd1.6 would limitbone damage, groups of five 6-10 week old, female Balb/c mice wereimmunized intraperitoneally with PBS (untreated control), anti-Gmd mAb1C11, anti-Amd mAb Amd1.6, or a combination (1C11+Amd1.6) at a totaldose of 40 mg/kg. Twenty-four hours later each mouse had insertedthrough its right tibia a pin contaminated with USA300 LAC::lux, abioluminescent CA-MRSA strain.

Bioluminescent imaging of all mice was performed on Days 0, 3, 5, 7, 10,and 14 using the Xenogen IVIS Spectrum imaging system (Caliper LifeSciences, Hopkinton, Mass.), and the peak BLI on Day 3 was quantified aspreviously described (Li et al., “Quantitative Mouse Model ofImplant-associated Osteomyelitis and the Kinetics of Microbial Growth,Osteolysis, and Humoral Immunity,” J Orthop Res 26:96-105 (2008), whichis hereby incorporated by reference in its entirety). A representativeBLI from each treatment group is illustrated in FIG. 7A, indicated thatbacterial load was present in each treatment group.

The resulting infection was allowed to progress for fourteen days whenthe animals were sacrificed and the infected tibiae were harvested foranalysis by microCT as previously described (Li et al., “Effects ofAntiresorptive Agents on Osteomyelitis: Novel Insights into thePathogenesis of Osteonecrosis of the Jaw,” Ann N Y Acad Sci 1192:84-94(2010), which is hereby incorporated by reference in its entirety). Inthe untreated control, bone lysis on both the medial and lateral sideswas extensive (FIG. 7B); Osteolytic Volume averaged over 0.4 mm³.Reductions in Osteolytic Volume were measured in all three groups ofantibody-treated mice (FIG. 7C). In one individual receiving thecombination therapy, the Osteolytic Volume was calculated to be 0,indicating a complete healing of the infected implant site. The effectof the combined antibody therapy in this individual is equivalent toboth a sterile pin and an infected pin that was cured with effectiveantibiotic therapy (i.e., gentamicin treatment in Li et al.,“Quantitative Mouse Model of Implant-Associated. Osteomyelitis and theKinetics of Microbial Growth, Osteolysis, and Humoral Immunity,” JOrthop Res 26:96--105 (2008), which is hereby incorporated by referencein its entirety). It is believed that this individual represents thefirst ever successful healing of an infected implant site in the absenceof antibiotic therapy.

Example 11—Passive Immunization with Anti-Amd mAb Amd1.6 SignificantlyReduces Bacterial Spread

The formation of abscesses is another indication of the severity ofinfection. The number of abscesses formed was measured in the same miceexamined in Example 10. Histological sections were stained with OrangeG/alcian blue (ABG/OH) which reveals abscesses as circular fields ofinflammatory host cells delimited by an unstained zone and, sometimes, adensely red staining nidus at its center. Typically, the nidus is theStaphylococcal abscess community (SAC); the inflammatory cells areneutrophils, mostly dead near the center and mostly alive near theperimeter and the unstained zone is a capsule formed from fibrin. In theuntreated mice multiple abscesses formed (FIG. 8A) with an average ofnearly 4.5 per tibia (FIG. 8C). In contrast mAb Amd1.6-treated miceaveraged only two abscesses as did those treated with the anti-Gmd mAb1C11 or with the combination (FIGS. 8B, 8C).

Example 12—Passive Immunization with Anti-Amd mAb Amd1.6 Alone or inCombination with Anti-Gmd 1C11 Promotes the Formation of SterileAbscesses and Accelerates Bone Healing

Detailed examination of the same histological sections presented in FIG.8B revealed unexpected findings. Consistently, intramedullarygram-stained abscesses were only found in tibiae of the PBS-treated mice(FIGS. 9A-B), while the lesions in the tibiae of the anti-Atl treatedmice were characteristic of sterile abscesses that did not containgram-positive bacteria (FIGS. 9C-H). Moreover, while the lesions in thetibiae of the placebo treated mice had clear histologic features ofStaphylococci abscess communities (SACs) (Cheng et al., “GeneticRequirements for Staphylococcus aureus Abscess Formation and Persistencein Host Tissues,” FASEB J 23(10):3393-3404 (2009); Cheng et al.,“Contribution of Coagulases Towards Staphylococcus aureus Disease andProtective Immunity,” PLoS Pathog 6(8):e1001036 (2010), each of which ishereby incorporated by reference in its entirety), no SACs were observedin the tibiae of anti-Atl treated mice (compare FIGS. 10A-B with FIGS.10C-H). Finally, and most surprisingly, it was discovered that combinedanti-Amd and anti-Gmd passive immunization not only clears the MRSAinfection (confirmed to be metabolically active on day 3; FIG. 7A) byday 14, but also allows for bone healing that has never been documentedto occur in this murine model of implant-associated osteomyelitis(compare FIGS. 11A-C). Specifically, osseus integration of the S. aureuscontaminated implant is documented in FIG. 11B, which displays a similarlevel of new bone formation around the pin and cortex as that observedin a sterile pin control (FIG. 11C). Using arginase-1-positive staining,the presence or absence of tissue healing M2 macrophages was alsoanalyzed. M2 macrophages, which are unable to enter the SAC in the tibiaof PBS treated mice (FIG. 11D), extensively invade the sterile abscessesin the tibia of combined anti-Amd and anti-Gmd treated mice tofacilitate classical tissue healing (FIG. 11E) (Murray and Wynn,“Protective and Pathogenic Functions of Macrophage Subsets,” Nat RevImmunol 11(11):723-737 (2011), which is hereby incorporated by referencein its entirety).

Example 13—Generation of Humanized Anti-Amd mAb Amd1.6

The variable regions of the light and heavy chains of the Amd1.6antibody will be PCR amplified using primers to permit cloning into thehuman antibody expression vectors described by Tiller et al. (“EfficientGeneration of Monoclonal Antibodies from Single Human B Cells by SingleCell RT-PCR and Expression Vector Cloning,” J Immunol. Methods329(1-2):112-24 (2008), which is hereby incorporated by reference in itsentirety). Plasmids containing the Amd1.6 light and heavy chain variableregions and human kappa and IgG1 constant regions will be prepared andco-transfected into HEK293 cells. After 3 days, the medium will beremoved from the cells and assayed for the presence of human IgG and forbinding to immobilized Amd protein by ELISA. Bound antibody will bedetected using a goat anti-Human IgG antibody coupled to horseradishperoxidase and 3,3′,5,5′ tetramethylbenzidene substrate.

To establish that the human:mouse chimeric Amd1.6 reacted with Amd aswell as the parental mouse Amd1.6, each will be tested for its abilityto inhibit the enzymatic activity of His-Amd.

The humanized Amd1.6 antibody can be utilized in a phase I clinicaltrial in elderly patients (>65 yrs) undergoing primary total jointreplacement. The humanized Amd1.6 antibody will be used alone and incombination with a humanized 1C11 anti-Gmd antibody as described in U.S.Patent Application Publ. No. 20130110249, which is hereby incorporatedby reference in its entirety.

Example 14—Generation of Humanized Anti-Amd mAb Amd2.1

The variable regions of the light and heavy chains of the Amd2.1antibody will be PCR amplified using primers to permit cloning into thehuman antibody expression vectors described by Tiller et al. (“EfficientGeneration of Monoclonal Antibodies from Single Human B Cells by SingleCell RT-PCR and Expression Vector Cloning,” J. Immunol. Methods329(1-2):112-24 (2008), which is hereby incorporated by reference in itsentirety). Plasmids containing the Amd2.1 light and heavy chain variableregions and human kappa and IgG1 constant regions will be prepared andco-transfected into HEK293 cells. After 3 days, the medium will beremoved from the cells and assayed for the presence of human IgG and forbinding to immobilized Amd protein by ELISA. Bound antibody will bedetected using a goat anti-Human IgG antibody coupled to horseradishperoxidase and 3,3′,5,5′ tetramethylbenzidene substrate.

To establish that the human:mouse chimeric Amd2.1 reacted with Amd aswell as the parental mouse Amd2.1, each will be tested for its abilityto inhibit the enzymatic activity of His-Amd.

The humanized Amd2.1 antibody can be utilized in a phase I clinicaltrial in elderly patients (>65 yrs) undergoing primary total jointreplacement. The humanized Amd2.1 antibody will be used alone and incombination with a humanized 1C11 anti-Gmd antibody as described in U.S.Patent Application Publ. No. 20130110249, which is hereby incorporatedby reference in its entirety.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. A monoclonal antibody, or binding portion thereof, that bindsspecifically to a Staphylococcus spp. autolysin consensusN-acetylmuramoyl-L-alanine amidase (Amd) catalytic domain and/or cellwall binding domain.
 2. The monoclonal antibody, or Amd binding portionthereof, according to claim 1, wherein the monoclonal antibody or Amdbinding portion inhibits in vivo growth of a Staphylococcus strainand/or biofilm establishment on metal, plastic and organic surfaces. 3.The monoclonal antibody, or Amd binding portion thereof, according toclaim 1, wherein the Staphylococcus strain comprises S. aureus, S.epidermidis, S. lugdunensis, S. saprophyticus, S. haemolyticus, S.caprae or S. simian.
 4. The monoclonal antibody, or Amd binding portionthereof, according to claim 1, wherein the Staphylococcus strain ismethicillin-resistant or vancomycin-resistant.
 5. The monoclonalantibody, or Amd binding portion thereof, according to claim 1, whereinthe antibody or Amd binding portion binds to an epitope of the Amdcatalytic domain.
 6. The monoclonal antibody, or Amd binding portionthereof, according to claim 1, wherein the antibody or Amd bindingportion binds to an epitope wholly or partly within the Amd R1 or R2cell wall binding domain.
 7. The monoclonal antibody, or Amd bindingportion thereof, according to claim 1, wherein the antibody or Amdbinding portion binds to the Amd catalytic domain or cell wall bindingdomain with an affinity greater than 10⁻⁸ M.
 8. The monoclonal antibody,or Amd binding portion thereof, according to claim 1, wherein theantibody or Amd binding portion binds to the Amd catalytic domain orcell wall binding domain with a K_(D) of about 1 to about 6 nM.
 9. Themonoclonal antibody, or Amd binding portion thereof, according to claim1, wherein the antibody or Amd binding portion comprises a V_(H) domaincomprising one of the following amino acid sequences:SEQ ID NO: 5 (Amd1.2):PELVKPGASVKMSCKASGYTFTSYIMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSTTAYMELSSLTSEDXAVYYCARLDGYYDCFDY WGQGTTLTVSSSEQ ID NO: 7 (Amd1.1):QQSGAELVKPGASVKLSCTASGFNIKDTYIHWVKQRPEQGLEWIGRIDPANGITNYDPKFQGRATITADTSSNIAYLQLTSLTSEGTAVYYCARGGYLSP YAMDYWGQGTSVTVSSSEQ ID NO: 9 (Amd1.5):QQSGAELVRPGALVKLSCKASGFNIQDYYLHWNKQRPEQGLEWIGWIDPENDNTVYDPKFRDRASLTADTFSNTAYLQLSGLTSEDTAVYYCARRDGITT ATRAMDYWGQGTSVTVSSSEQ ID NO: 11 (Amd1.6):QSGTVLARPGTSVKMSCKASGYSFTNYWMHWVRQRPGQGLEWIGSIYPGNSDTTYNQKFKDKAKLTAVTSASTAYMELSSLTNEDSAVYYCTGDDYSRFS YWGQGTLVTVSASEQ ID NO: 13 (Amd1.7):QQSGPELVKPGASVKISCKASGYTFTDYNMHWVKQSHGKSLEWIGYIFPYNGDTDYNQKFKNKATLTVDNSSSTAYMDLRSLTSEDSAVYYCSRWGSYFD YWGQGTTLTVSSSEQ ID NO: 15 (Amd1.9):VESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPKKSLEWVASITSGGSAYYPDSVKGRFTISRDNARNILNLQMSSLRSEDTAMYYCARDDGYFDY WGQGTTLTVSSSEQ ID NO: 17 (Amd1.11):QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLEWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLLINNLKNEDTATYFCARRD GYFDAMDYWGQGTSVTVSSSEQ ID NO: 19 (Amd1.12):QQSGAELVRPGTSVKVSCKTSGYAFTNYLIEWVNQRPGQGLEWIGVINPGSGGTNYNEKFKAKATLTADKSSSTAYMQLSSLTSDDSAVYFCARSERGYY GNYGAMDYWGQGTSVTVSSSEQ ID NO: 21 (Amd1.13):QQPGPELVKPGASLKISCKASGYSFSSSWMNWVKQRPGQGLEWIGRIYPVDGDTNYNGKFKGKATLTTDKSSSTAYMQLSSLTSVDSAVYFCARTGPYAM DYWGRGTSVTVSSSEQ ID NO: 23 (Amd1.16):GAELVRPGSSVKISCKASGYTFSTYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCARSMVTNYYFA MDYWGQGTSVTVSSSEQ ID NO: 25 (Amd1.17):GGLVKPGGSLKLSCAASGFTFSDYYMYWVRQTPEKKLEWVATISDGGSYTYYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYCVRGLLGFDYWGQ GTTLTVSSand/or wherein the antibody or Amd binding portioncomprises a V_(L) domain comprising one of thefollowing amino acid sequences: SEQ ID NO: 33 (Amd1.1):ENVLTQSPAIMSASLGEKVTMTCRASSSVNYMFWFQQKSDASPKLWIYYTSNLAPGVPARFSGSGSGNSYSLTISSMEGEDAATYYCQEFTSFPYTFG SEQ ID NO: 35 (Amd1.2):DIVLTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPQYTFSEQ ID NO: 37 (Amd1.6):SIVMTQTPKFLLVSAGDRLTITCKASQSVSNDVAWYQQKPGQSPKLLIYYTSNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYNSPWTFGG GTKSEQ ID NO: 39 (Amd1.7):SIVMTQTPKFLLVSAGDRLTITCKASQSVSNDVAWYQQKPGQSPKLLIYYTSNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYNSPWTFGG GTKSEQ ID NO: 41 (Amd1.8):DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQRSHESPRLLIKYVSQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPYTFGSEQ ID NO: 43 (Amd1.9):DIQMTQSPASLSVSVGETVTITCRTSENIFSNFAWYQQQPGKSPQLLVYGATNLADGVPSRFSGSGSGTQYSLKITSLQSEDFGSYYCQHFWGSPWTFSEQ ID NO: 45 (Amd1.10):QIVLTQSPALMSASPGEKVTMTCSASSSVSYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPYTFGSEQ ID NO: 47 (Amd1.11):DILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPALTFGSEQ ID NO: 49 (Amd1.12):DIQMTQSPASLSASVGDTVTITCRASENIYSYLAWYQQKQGKSPQLLVYNAKTFAEGVRSRFSGSGSGTQFSLQITSLQPEDFGSYYCQHHYGSPYTFSEQ ID NO: 51 (Amd1.13):DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLISWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSY PFTFGSEQ ID NO: 53 (Amd1.15):DIAMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVRDRFXGSRCGTDFTFPISSVQGEDLAVYYCQQHYSIHSRSSEQ ID NO: 55 (Amd1.17):DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP WTFGGGT,


10. The monoclonal antibody, or Amd binding portion thereof, accordingto claim 1, wherein the antibody or Amd binding portion comprises aV_(H) domain comprising one of the following amino acid sequences:SEQ ID NO: 27 (Amd 2.1):GFVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEMRLEWVASISSGGSXTYYPDSVMGRFTISRDNARNILNLQMSSLRSEDTAMYYCARVGLYYDYYYSM DYWGQGTSVTVSSSEQ ID NO: 29 (Amd 2.2):ESGPELVKPGASVKISCKASGYTFTDYNMHWVRQSHGKSLEWIGYIYPYNGGTGYNQKFKSKATLTVDNSSSTAYMELRSLTSEDSAVYYCAREDGYYGY GDYWGQGTTLTGSSSEQ ID NO: 31 (Amd 2.4):QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASAAYLQINNLKNEDTATYFCARDY DGYYYYAMDYWGQGTSVTVSSand/or wherein the antibody or Amd binding portioncomprises a V_(L) domain comprising one of thefollowing amino acid sequences: SEQ ID NO: 57 (Amd 2.1):DISMTQSPSSLTVTAGEKVTMSCKSSQSLLYSGNQKNYLTWYQQKPGQPPKMLIYWASTRESGVPDRFTGSGSGTHFTLTISSVQAEDLAIYYCQNDYSY PVTFGAGTKLELKSEQ ID NO: 59 (Amd 2.2):EIVLIQSPAITAASLGQKVTITCSASSSVNYMHWYQQKSGTSPKPWIYEISKLASGVPARFSGSGSGTSYSLTISSMEAEDAAIYYCQQWNYPLITFGAG TKLELKSEQ ID NO: 61 (Amd 2.4):ENALTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSSMSPKLWIYDTSKLASGVPGRFSGSGSGNSYSLTISSMEAEEVATYYCFQGSGFPVHVRRG DQVGNKTSEQ ID NO: 63 (Amd 2.5):DIQMTQSPASLSASVGETITITCRASGNIHNYLAWYQQKQGKSPHLLVFHARSLADGVPSRFSGSGSGTQYSLNINSLQPEDFGIYYCQHFWYTPYTFGG GTKLEIK.


11. The monoclonal antibody, or Amd binding portion thereof, accordingto claim 1, wherein the monoclonal antibody or Amd binding portion ispartially humanized or fully human.
 12. The monoclonal antibody, or Amdbinding portion thereof, according to claim 11, wherein the humanizedmonoclonal antibody is IgG1, IgG2, IgG3, or IgG4 class.
 13. The Amdbinding portion according to claim
 1. 14. The Amd binding portionaccording to claim 13, wherein the Amd binding portion comprises a Fabfragment, Fv fragment, single-chain antibody, a V_(H) domain, or a V_(L)domain. 15-31. (canceled)
 32. A cell line that expresses a monoclonalantibody according to claim 1 or an Amd binding portion thereof. 33-34.(canceled)
 35. A pharmaceutical composition comprising a carrier and oneor more monoclonal antibodies according to claim 1 or one or more Amdbinding portions thereof. 36-44. (canceled)
 45. A method of introducingan orthopedic implant, graft or medical device into a patientcomprising: administering to a patient in need of an orthopedic implant,graft or medical device an effective amount of a monoclonal antibodyaccording to claim 1, or one or more Amd binding portions thereof;introducing the orthopedic implant, graft or medical device into thepatient. 46-68. (canceled)
 69. A method of treating or preventing aStaphylococcus infection comprising: administering to a patientsusceptible to or having a Staphylococcus infection an effective amountof a monoclonal antibody according to claim 1, or one or more Amdbinding portions thereof. 70-85. (canceled)
 86. A method of treatingosteomyelitis comprising: administering to a patient having aStaphylococcus bone or joint infection an effective amount of amonoclonal antibody according to claim 1, or thereof. 87-101. (canceled)102. A method of determining presence of Staphylococcus in a sample, themethod comprising: exposing a sample to a monoclonal antibody accordingto claim 1 or an Amd binding portion thereof; and detecting whether animmune complex forms between the monoclonal antibody or binding portionand Staphylococcus or a Staphylococcus amidase present in the sample,whereby presence of the immune complex after said exposing indicates thepresence of Staphylococcus in the sample. 103-107. (canceled)